Linfoma Diffuso a Grandi Cellule B e Grey Zone

Corsi della scuola nazionale di anatomia patologica La patologia neoplastica del sistema emolinfopoietico: attualità e prospettive. Bologna, 10-12 dicembre 2014 Linfoma Diffuso a Grandi Cellule B e Grey Zone Facchetti Fabio Brescia Giovedì 11 dicembre 2014 10.45-12.45

Histiocytic Medullary Reticulosis (Scott & Robb-Smitt)(1939) Histiocytic Reticulosis Malignant Reticulosis Histiocytic Leukemia Malignant Histiocytosis (Rappaport)(1966) Cancer 1990 La maggior parte delle neoplasie un tempo definite istiocitosa maligna sono Linfomi NH ad alto grado (B >>T)

DLBCL De-novo Secondari CLL (Richter) MALT Follicolare

CD5 Richter

Cosa e come discuteremo Alcune forme particolari di DLBCL e problematiche correlate Linfoma intravascolare Linfomi EBV+ Linfomi HHV8+ Linfoma B ALK+ Linfoma plasmablastico (Prof. Leoncini) DLBCL NOS (la variante più comune) Caratteristiche generali Il problema della stratificazione prognostica secondo espressione genica e surrogati fenotipici DLBCL e borderline con altre neoplasie THR-DLBCL HL-NLP Med DLBCL CHL ( grey-zone lymphoma in senso stretto) DLBCL BL (da un concetto di borderline DLBCL-BL a una nuova possibilità di stratificare prognosticamente i DLBCL- NOS)

Intravascular Large B-cell lymphoma (WHO 2008) Intravascular Large B-cell lymphoma (Primary cutaneous diffuse large B-cell lymphoma, other) (WHO-EORTC 2005) Synonym: Angiotropic large cell lymphoma IVLBCL is a rare type of extranodal large B- cell lymphoma characterized by the selective growth of lymphoma cells within the lumina of vessels, with exception of larger arteries and veins (WHO, 2008).

Blood vessels often dilated and containing large lymphoid cells

Possible fibrin deposition and vascular occlusion

Intravascular blasts are loose or densely packed Scattered extravascular atypical cells can be present

CD19+, CD20+, CD22+, CD79a+ Monotypic sig+ (frozen) Usually non-gc-type (BCL6- CD10-MUM1+) BCL2+ CD5+ (29% 75%) PSA+ in rare cases EBV and HHV8: negative BCR clonally rearranged No consistent cytogenetic anomalies CD20

CD19+, CD20+, CD22+, CD79a+ Monotypic sig+ (frozen) Usually non-gc-type (BCL6- CD10-MUM1+) BCL2+ CD5+ (29% 75%) PSA+ in rare cases EBV and HHV8: negative BCR clonally rearranged No consistent cytogenetic anomalies Blood vessels CD31+CD34+ Podoplanin-Lyve1- CD34

Data indicate a defective expression of adhesion molecules (esp. CD29/b1 integrin and CD54/ICAM1) on tumor cells. Ponzoni et al. Hum Pathol 2000;31:220-6 Krober et al. X EAH Meeting, London, 2000 Kinoshita et al. Brain Tum Pathol 2008; 25:73-8 CD29 Courtesy by M. Ponzoni CD29 CD54

INTRAVASCULAR B-CELL LYMPHOMA CLINICO-PATHOLOGICAL FEATURES 1. Two clinical subtypes: Classical and Asian 2. Skin manifestations of IVL 3. The cutaneous variant

IVL B-cell type Clinical subtypes Classical More common in Western countries M:F~ 1 Median age: 70 (34-90) Systemic, with predominant CNS (39%) and Skin (39%) involvement No tumor masses; very rare lymphadenopathy B-symptoms common (55%) Abnormal laboratory findings frequent (anemia, thrombocytopenia, LDH, hypoalbuminemia) Median survival: 12 mths 3-year OS: 32% Asian More common in Far-eastern countries M:F~ 1 Median age: 67 (41-85) Systemic, with multiorgan failure and frequent hemophagocytic syndrome. Less frequent CNS (27%) and skin (15%) involvement More frequent hematopoietic tissue involvement (esp.: spleen, P.B., B.M.) B-symptoms common (76%) Abnormal laboratory findings very frequent (esp: trombocytopenia, hypoalbuminemia) Median survival: 8 mths 3-year OS: 18% The oncologist's "great imitator A lymphoma is rarely suspected

Data from 224 patients with IVL Skin lesions: 90/224 (40.2%) Thigh (41%), Leg (35%), Trunk (31%) Nodules and/or plaques (49%) Macules (22.5%) (red or blue to livid colour) Telangiectases 20% Associated: edema (27.5%) and pain (24%) No clearcut lymphomatous appearance, but often misinterpreted as: Thrombophlebitis Erythema nodosum Erysipelas Livedo racemosa Vasculitis Purpura Courtesy H. Kutzner

Br J Hematol 2004; 127:173-183 CUTANEOUS COMPARED TO THE CLASSICAL VARIANT Females Younger (median 59 vs. 72) Normal hematological values, but similar LDH, anemia, ESR anomalies Less frequent B-symptoms No bone marrow involvement Better survival, especially when single lesions (3-year $: 56%) p= 0.007 Br J Dermatol 2007; 157:16-25 Review of 224 patients (97 articles) Disease restricted to the skin prognosis favourable (85% vs. 10% fatal outcome).

10 cases reported 5M, 5F Kobayashi et al. Mean age: 66.5 yr (52-82) IVL colonizing a preexisting hemangioma (6) or manifesting as eruptive hemangiomas (4) Small (cherry) or medium size When multiple, not all hemangiomas are involved by IVL Extracutaneous IVL: 8/10 Follow-up 3 DOD (1-12-23 months after dia.) 1 recurrence, alive after BMT 6 alive after CT Cerroni et al.

López-Gil F, Roura M, Umbert I, Umbert P. Malignant proliferative angioendotheliomatosis or angiotropic lymphoma associated with a soft-tissue lymphoma. J Am Acad Dermatol 1992;26:101-4. Cerroni L, Massone C, Kutzner H, Mentzel T, Umbert P, Kerl H. Intravascular large T-cell or NK-cell lymphoma: a rare variant of intravascular large cell lymphoma with frequent cytotoxic phenotype and association with Epstein-Barr virus infection. Am J Surg Pathol 2008;32:891-8. IVL T/NK-cell type Extremely uncommon Not included (yet) in any classification Compared with IVL-B Younger: median: 58 vs. 70 M>F (2:1) Similar cutaneous clinical presentation Survival: very aggressive (but better survival if lesions limited to a single organ, including the skin) PHENOTYPE T/NK (=nasal-type), CD2 + CD3 + CD5 - CD56 + bf1 - EBV + T (=PTCL-nos) T (=ALCL, CD4 + CD30 + ALK - ) GENOTYPE TCR rearranged in 1/3 of cases

Courtesy Dr. Rui Bajanca

TIA1 CD56 bf1 EBER

EBER EBER??? T/NK nasal-type (intravascular) T/NK nasal-type (angiocentric)

Bryant A, et al

T-cell phenotype CD30+/- High Ki67 TCR: germline CD30 Intralymphatic 3 cases, F-up negative Ki67 Podoplanin

Female, 69 Solitary pyogenic granuloma on the arm No other abnormalities Alive and well after 30 months

CD3 bf1 CD30 Ki67 A Podoplanin CD4 FoxP3 F B C CD8 CD25 CCR7 D E A. Intralymphatic B. T-cell phenotype C. CD4+ CD8- D. High proliferation index, modest CD30 expression E. TCR: polyclonal F. Effector-memory T-regs phenotype G. Migratory phenotype G?? Locally activated/proliferating T- helper cells, with effectormemory T-reg phenotype, migrating to draining lymph nodes

Cutaneous intravascular (intralymphatic) atypical blastic T-cell proliferations mimicking IV lymphomas Author Clinical Phenotype Genotype Vessels Follow-up Ardighieri et al. (JCP, 2009) Right arm 69, F Solitary 10mm lesion CD: pyogenic granuloma CD3+CD4+ CD30-/+ Lymphatic NED after 30 months TCR: polyclonal Baum et al. (JCP, 2009) Right forearm 17, M Appeared 7-8 weeks after trauma CD: pyogenic granuloma CD3+CD4+ (60%), CD8+ (10%) CD30+ Vascular (CD31+) NED after 8 months TCR: oligoclonal Cesinaro et al, (JCP, 2010) Right flank 56, F Rapid development of a pyogenic granuloma n.a. Lymphatic NED after 15 months Fernandez- Figueras MT (AJD, 2013) Neck 17, F Long standing hemangioma, ulceration and exophytic growth after trauma, no tendency to heal spontaneously CD3+CD4+ CD30-/+ TCR: n.a. Lymphatic NED after 12 months

Benign Intralymphatic Blast Proliferations Differential Intravascular Lymphomas IVL, large B-cell type IVL, T/NK type 1. More loose intravascular cells 2. Vascular and not lymphatic 3. No/minimal extravascular component 4. Usually B (may occur in hemangiomas) 5. The rare T/NK are EBV+

Descrivono uno spettro di lesioni T CD30+ intralinfatiche, che comprendono: Unilesionali (2) Papulosi Linfomatoide (3) ALCL ALK- (6), con recidive locali, ma clinicamente indolenti ALCL ALK+ (1) aggressivo

EBV DEMONSTRATION IHC LMP1 (Latent Membrane Protein 1) ISH EBER (EBV encoded RNA) Not equivalent!! LMP 1 EBER 1-2 Latency Phase I II III

Testis, NK/T cell lymphoma LMP1 EBER

EBER caveat! EBV 1-5% di tutti I tessuti linfoidi (esp. tonsille) contengono sparse cellule positive La semplice presenza di rare cellule EBV+ non indica che una eventuale alterazione morfologica del tessuto linfoide sia EBVcorrelata Numerose cellule EBV+ possono indicare anche quadri reattivi: Mononucleosi infettiva Linfoadenite EBV+ cronica Caratteristiche morfologiche e fenotipiche simili Quadro clinico differente Riattivazione virale (specie intrafollicolare) Ulcere muco-cutanee associate o meno a stati di immunodeficit

*: residui follicoli con mantello (espansione non invasiva ) * * * Mononucleosi Infettiva Espansione paracorticale Pleomorfismo cellulare Cellule piccole Cellule grandi (n variabile) Rare cellule medie Atipia scarsa, ma RS-like cells possibili

SPETTRO DI DIFFERENZIAZIONE PLASMACELLULARE (M.I.)

CD5 CD8 CD20 Eterogeneità fenotipica dei blasti (T e B); spesso prevalenza di CD8 (fase di eliminazione di infezioni virali)

Reazione paracorticale diffusa immunoblastica Criteri di benignità k l Non destruente (follicoli residui, non massa espansiva) Eterogeneità fenotipica Non clonalità catene leggere

Reazione paracorticale diffusa immunoblastica Criteri di benignità Non destruente (follicoli residui, non massa espansiva) EBER Eterogeneità fenotipica Non clonalità catene leggere Clinica!! Età: giovane? Sede: tonsille; laterocervicale? Angina? Febbre?

35 male Disseminated lymphadenopathy CD: lymphoma

EBER EBV lymphadenitis Serum EBV++++ Spontaneous regression LMP1

Riattivazione virale con numerose cellule centrofollicolari EBV+

2010 17 nessuna associazione (età m/na 80) 9 terapia immunosoppress. per varie mal. autoimmuni (età m/na 69) Lenta insorgenza ulcere anche ampie Mucosa oro-faringea (16), tratto GI (7), cute (6) Non segni sistemici Evoluzione indolente con risoluzione spontanea o dopo sospensione di terapia immunosoppressiva (rari CHT o RT per diagnosi di Linfoma) 2014 7 terapia immunosoppress. per trapianto organo solido (età m/na 61) Mucosa orale o tratto GI Non segni sistemici Risoluzione rapida dopo riduzione terapia immunosoppressiva (2 dec. per sepsi)

ULCERE MUCO-CUTANEE EBV+ Ulcere ben delimitate Infiltrato polimorfo con variabile n di grandi cellule atipiche, anche R-S-like Occasionale angiotropismo; necrosi Cellule atipiche di natura B, con: espressione parzialmente difettiva di mks B (CD20, Pax5), spesso IRF4+ e CD30+ EBV+ Cellule reattive specie T, pleomorfe (prevalenza CD8+) Altre componenti infiammatorie, talora prominenti (PC, Istio, Eos,..) Clonalità (due studi citati) IgH: 10/33 (7+3) TCR : 6/33 (6+0) EBV circolante (studio Hart) Valori molto bassi o negativi ( casi PTLD!!!)

F, 70 Ulcera base lingua

EBER CD20 + EBER

IgH clonale (identica su due campioni distinti)

M, 61 RCU con vari trattamenti Ulcera mucosa e stenosi concentrica a 25 cm dal margine anale

EBER CD20- CD45- Pax5+ IRF4+ CD138- CD30+ CD15+ (rari) Completa risoluzione della lesione

EBV+ DLBCL DLBCL of the elderly Lymphomatoid Granulomatosis DLBCL in chronic inflammation Associazioni Sedi Evoluzione Età >50 anni Specie asiatico (8%-10% di tutti i DLBCL), ma anche in P. Occidentali (3%-4%) Adulti (mediana 57; 24-76) Rari casi con associata immunodeficienza Forma più frequente associata a patologia pleurica cronica (PAL) Altre: ulcere cutanee croniche, osteomielite cronica, protesi, 70% extranodali (cute, polmoni, tonsille, GI,..) 30% nodale Extranodale (polmoni 90%; cute, fegato, reni 10%; ) Tipicamente pleuro-polmonare (PAL = pyothoraxassociated lymphoma) Aggressivo, specie in >70 anni Prognosi dipendente dal grado (molto aggressiva LyG G3) Aggressivo

DLBCL elderly type Quadri pleomorfi o monomorfi Spesso necrosi EBV+ nella maggior parte delle cellule atipiche

DLBCL elderly type EBER

90% coinvolgimento polmonare, con: Noduli bilaterali, spesso periferici, anche cavitati Possibili quadri reticolo-nodulari

Lesioni cerebrali Lesioni cutanee Lesioni renali

Granulomatosi Linfomatoide Infiltrato polimorfo Cellule reattive (>> T CD4+; Mf, PC. No PMN/EOS!!) Cellule B grandi (EBV+)(CD20+, CD30 var., CD15-) Angiocentrico/angiodistruttivo Necrosi (estensione variabile) Non granulomi!! (il termine granulomatosi è del tutto improprio)

10727-07

CD3 CD68 CD20 EBV (EBER)

CD20 CD68

CD20 EBER

Granulomatosi Linfomatoide Gradazione (1-2-3) in relazione al n di grandi cellule atipiche EBV+ Grado I Grado II Grado III Grandi cellule EBV+ 0-5 / hpf 5-20 50 /hpf >50 /hpf Necrosi Componente reattiva PROGNOSI Dipende dalla estensione delle lesioni e dal Grado Grado I-II possono regredire (specie se in forme localizzate) risposta a INF-alfa evoluzione in Grado III nel 30-60% dei casi Grado III raramente autoregrediscono (solo le forme limitate) linfoma aggressivo, trattato come un linfoma B ad alto grado (R- CHOP), ha una sopravvivenza mediana <2 anni

WHO, 2008 Pyothorax Associated Lymphoma (PAL)

Pyothorax Associated Lymphoma Specie Orientali (Japan) Francia, Italia Maschi Grandi masse pleuriche estendentisi alla parete toracica e/o al parenchima polmonare, ma raramente associate a versamento intracavitario Diffusione per contiguità Rara disseminazione Altamente aggressivo

Pyothorax Associated Lymphoma EBV+ HHV8- In genere CD20+ Alcuni casi plasmablastici (CD20- CD138+ IRF4+) Possono CD30+ Alcuni casi difettivi B Molto raramente bifeno- e bigenotipico (anche CD3+ CD4+, con riarrangiamento clonale del TCR-AB)

CD138 EBER CD20

CD138 EBER

Processi Linfoproliferativi PEL (Primary di Interesse Effusion Bronco-Pneumologico Lymphoma) Epidemiology similar to Kaposi Sarcoma Males Young HIV+ (esp. homosexual) Eldery non-hiv+ Malignant effusion of one or more cavities No/minimal tumor masses 20% extra-serosal extension Very aggressive (median $: 5 months) Exceptionally response to HAART

Sospettarlo quando: Citologico francamente maligno Molto anaplastico Molto apoptotico

CD20 CD30 CD45+ CD20, Pax5 spesso negativi CD138+ CD30+ JH riarrangiato Possibile aberrante espressione di marcatori T HHV8+ (elemento definente ) EBV spesso associato (specie nei p.ti HIV+) ORF73

ASCITE

ASCITE HHV8

HIV+ Liquor

Condizione di base più frequente Presentazione PEL Immunodeficit (HIV) Versamento senza massa PAL Piotorace Massa, spesso senza versamento Virus associato/i HHV8 (+/- EBV) EBV EBV latenza Tipo I (LMP1-) Tipo III (LMP1+) Fenotipo B Spesso difettivo Solitamente non difettivo Decorso Molto aggressivo Molto aggressivo

Kaposi s sarcoma-associated herpesvirus-like DNA sequences in multicentric Castleman s disease. Soulier et al. Blood, 1995;86:1276-1280 HHV-8 is associated with plasmablastic variant of Castleman disease that is linked to HHV-8 positive plasmablastic lymphoma. Dupin et al. Blood, 2000;95:1406-1412 HHV8+ MULTICENTRIC CASTLEMAN DISEASE M.C.D. is HHV8+ in 100% HIV+ and in 40% HIV- patients Systemic lymphadenopathy Epato-splenomegaly Systemic symptoms Hypergammaglobulinemia Thrombocytopenia Anemia Autoimmunity Frequently KS associated PLASMABLASTIC LYMPHOMA ARISING IN HHV8+ MCD HIV+ with profound immunodeficiency Lymph node and spleen markedly enlarged Extension to other sites Leukemia possible Often KS manifestly associated Highly aggressive, with median survival of few months

HHV8+ MULTICENTRIC CASTLEMAN DISEASE

HHV8 (ORF73)

PLASMABLASTIC LYMPHOMA

HHV8 (ORF73)

CD20 MUM1 Kappa PLASMABLASTS in MCD HHV8+ EBV- CD20- or CD20-/+ CD138- or CD138+ IRF4/MUM1+ IgM/l (naive) IG genes are unmutated Early stages polyclonal, then monoclonal Lambda

Neoplasie ALK+ (traslocazioni, mutazioni, amplificazioni,?) ALCL, ALK+ DLBCL Rabdomiosarcoma (45% A, 15% E) Tumore Miofibroblastico Infiammatorio Carcinoma NSC polmone Neuroblastoma Altri (carcinoma esofago, rene, mammella, colon, tiroide) Variante di Nevo di Spitz Rabdomiosarcoma alveolare

Neoplasie ALK+ (traslocazioni, mutazioni, amplificazioni,?) ALCL, ALK+ DLBCL Rabdomiosarcoma (45% A, 15% E) Tumore Miofibroblastico Infiammatorio Carcinoma NSC polmone Neuroblastoma Altri (carcinoma esofago, rene, mammella, colon, tiroide) Variante di Nevo di Spitz Tumore Miofibroblastico Infiammatorio

Anaplastic LCL T/null, ALK+ e ALK-(Stein) Anaplastic LCL B, ALK+ (full-lenght ALK?)(Delsol) ALCL B-cell type does not exist Anaplastic LCL B, ALK+ 1985 1997 2003 Blood 1 st October 2003; vol. 102 2568-73 2638-41 2642-44 11 cases Age variable; 9/2 M/F Sinusoidal growth Plasmablastic morphology CD20 - CD79a -/+ CD138 + CD30-9 CLTC/ALK 2 NPM/ALK Aggressive in 6/11 cases

Cosa e come discuteremo Alcune forme particolari di DLBCL e problematiche correlate Linfoma intravascolare Linfomi EBV+ Linfomi HHV8+ Linfoma B ALK+ Linfoma plasmablastico (Prof. Leoncini) DLBCL NOS (la variante più comune dei DLBCL; 30% di tutti i LNH) Caratteristiche generali Il problema della stratificazione prognostica secondo espressione genica e surrogati fenotipici DLBCL e borderline con altre neoplasie THR-DLBCL HL-NLP Med DLBCL CHL ( grey-zone lymphoma in senso stretto) DLBCL BL (da un concetto di borderline DLBCL-BL a una nuova possibilità di stratificare prognosticamente i DLBCL- NOS)

Lai R., et al. Sinusoidal CD30-positive Large B-cell lymphoma: a morphologic mimic of Anaplastic Large Cell Lymphoma. Mod Pathol 2000; 13:223-228.

CD20 CD30 CD20+ CD30+ ALK- ALK

Categoria eterogenea per: Morfologia e fenotipo Alterazioni genetiche Risposta alla terapia

DLBCL-NOS (Dr. G. Rossi, Ematologia, Brescia) Terapia modulata in base alla prognosi. Score più utilizzato: IPI (Indice Prognostico Internazionale) 4 gruppi prognostici in base a età, PS, LDH, stadio e n sedi extranodali. Molti tentativi di aggiungere parametri prognosticamente significativi a queste semplici variabili cliniche, tuttora non universalmente accettati.

DLBCL-NOS (Dr. G. Rossi, Ematologia, Brescia) Terapia d elezione: R-CHOP (R= rituximab [AbMo anti CD20]; C ciclofosfamide H adriamicina, O vincristina, P prednisone). 6-8 cicli, riducibili in stadi iniziali, associando radioterapia. Tuttora non chiaro ruolo di trapianto staminali autologhe in pazienti a prognosi sfavorevole come prima linea di terapia. Trapianto autologo comunque terapia di elezione dopo recidiva.

DLBCL-NOS Note di terapia (Dr. G. Rossi, Ematologia, Brescia) Sopravvivenza a lungo termine: 70% circa Pazienti in remissione per due anni dopo la prima linea terapeutica hanno una aspettativa di vita simile a quella di un individuo normale I tentativi di migliorare questi risultati prevedono l aggiunta di farmaci non chemioterapici: lenalidomide ad azione immunomodulante bortezomib ad azione sul pathway di NFkB nuovi anticorpi monoclonali (GA-101: anti CD20 con maggiore azione ADCC e apoptotica rispetto a rituximab) piccole molecole come gli inibitori della via di segnale del BCR (ibrutinib) o inibitori di bcl-2.

Alla ricerca di surrogati immunoistochimici della classificazione GEP dei DLBCL-NOS Dal 2004 sono stati sviluppati diversi algoritmi immunoistochimici basati sulla espressione di 3-5 antigeni con l obiettivo di riconoscere le due principali categorie prognostiche (GCB e non- GCB o ABC) come definite dal GEP Questi algoritmi si basano sulla espressione di pochi antigeni comuni quali: CD10, BCL6, MUM1/IRF4, CGET1, FoxP1 (BCL2, LMO2) L iter classificativo e i cut-off di positività variano nei diversi algoritmi Il significato prognostico e la correlazione con GEP spesso non sono stati confermati quando applicati da altri Autori o in studi meta-analitici

TISSUE MICROARRAY DA 36 PAZIENTI CON DLBCL 8 LABORATORI, 8 ANTICORPI (CD5, CD10, CD20, BCL2, BCL6, MUM1, MIB1, HLADR)

DLBCL-NOS Note di terapia (Dr. G. Rossi, Ematologia, Brescia) La conoscenza del sottotipo genico del DLBCL, GC-like o ABC, non è ancora entrata nella pratica clinica Tuttavia sembra che l azione dei nuovi farmaci si eserciti selettivamente su uno dei due sottotipi (ABC sia per lenalidomide che per bortezomib e ibrutinib). Per questo sono in corso protocolli clinici che valutano queste associazioni, classificando dal p.d.v. molecolare il linfoma prima dell inizio della terapia.

Nodular lymphocyte-predominant Hodgkin Lymphoma (NLPHL) Most often between 30-50 years, but the most frequent HL subtype in pre-adolescent children; strong prevalence in males Generally low-stage disease Clinical course is indolent with high rate of cure Despite late relapses may occur, they are typically localized and still responsive to therapy. T-cell/Histiocyte rich Diffuse Large B-cell Lymphoma (THRLBCL) Middle aged patients, predominantly males High-stage presentation (multiple lymph nodes, liver, spleen; often bone marrow involvement) Response to conventional chemotherapy is poor

Nodular Lymphocyte Predominance Hodgkin Lymphoma (NLPHL) T-cell/Histiocyte rich Diffuse Large B-cell Lymphoma (THRLBCL) In their classical presentation they show totally different histological features

NLPHL CD20 CD21

CD20 CD3

LNH-B-GC, ricco in T/istiociti Architettura: pattern diffuso <10% di cellule neoplastiche (WHO) Non devono coesistere numerosi linfociti B di piccola-media taglia Non aggregati di grandi cellule B

SFONDO REATTIVO (in questo caso linfocitico) e sparse cellule atipiche grandi

SFONDO REATTIVO (in questo caso istiocitico) e sparse cellule atipiche grandi

Non inusuale identificare RS-likE cells

THRLBCL

LNH-B-GC, ricco in T/istiociti CD20 <10% di cellule neoplastiche Intensità marcata

The boundary between Nodular Lymphocyte Predominance Hodgkin Lymphoma (NLPHL) and T-cell/Histiocyte rich Diffuse Large B-cell Lymphoma (THRLBCL). NLPHL may show: single/multiple nodules diffuse areas with morphological features similar to THRLBCL

CD23 (FDC) CD3 CD20

NLPHL (± THRLBCL-like areas) versus THRLBCL 1. Tumor cell phenotype is almost identical Incostant (and practically poorly applicable) differences in expression of markers such as LSP1, PU.1, FREB, MUM1, Molecular features in NLPHL and THRLBCL Similarities Rearranged, mutated IGH genes with ongoing mutations Frequent partial gain of 4q and losses of 19/19p Differences BCL6 translocation (>> in NLPHL) In NLPHL higher number of genomic imbalances (average of 4.7 in THRLBCL versus 10.8 in NLPHL) and distribution (usually 1 5 in THRLBCL versus 6 22 in NLPHL) GEP analysis show more similarities than differences between tumor cells in NLPHL and THRLBCL

Brune V, et al. Origin and pathogenesis of nodular lymphocyte-predominant Hodgkin lymphoma as revealed by global gene expression analysis. J Exp Med 2005; 10: 2251-2268 (3364 genes) Genome-wide expression study of microdissected L &H lymphoma cells in comparison to normal and other malignant B cells L&H cells show a surprisingly high similarity to the tumor cells of THRLBCL Only 42 genes significantly differ (overexpressed in L&H cells)

GEP on microdissected tumor cells Both in unsupervised and supervised analyses the lymphoma cells did not cluster according to their entity. There are no clear and consistent differences in the gene expression of the tumor cells of NLPHL, THRLBCL-like NLPHL and THRLBCL. 2013

NLPHL (± THRLBCL-like areas) versus THRLBCL 2. Tumor-associated cells differ FHT-cells more abundant in NLPHL TIA1+/CD8+ T-cells more abundant in THRLBCL The CD57+/TIA1+ ratio is high in NLPHL, low in THRLBCL

NLPHL Rosetting by Follicular Helper T Cells CD4 PD1 BCL6 CXCL13 CD57 ICOS PD1 CXCL13 BCL6 CD57

NLPHL (diffuse area) PD1 THRLBCL

NLPHL (diffuse area) CD57 THRLBCL

THRDLBC CD3 >> CD8 (TIA1+)

NLPHL (diffuse area) TIA1 THRLBCL

Marked differences in tumor microenvironment NLPHL: very similar to a lymph node characterized by follicular hyperplasia THRLBCL: up-regulation of CCL8, interferon-γ, indoleamine 2,3 dioxygenase (IDO), VSIG4 and Toll-like receptors. recruitment and activation of T cells, macrophages and dendritic cells, innate immunity and tumor tolerogenic immune response

Primary Component Analysis 2013

THRLBCL NLPHL CD163

Common Progenitor (4p gain, 19p/19 loss ) OTHERS MICROENVIRONMENT NLPHL Immunogenic? Tolerogenic THRLBCL

NLPHL with THRLBCL-like areas Clinical significance? The presence of TCRBCL-like patterns may predict recurrence, but it is not associated with a more aggressive behavior Fan Z, Am J Surg Pathol 2003 Boudova L, Blood 2003 Terminology to be used: Boudova L, Blood 2003 NLPHL with THRLBCL-like areas instead of THRLBCL (WHO, 2008).

THRLCL NLPHL? with THRLBCL-like areas

NLPHL with THRLBCL-like areas versus autentic THRLBCL Consider NLPHL: Any nodule (!) @-CD20 and @-FDC markers useful to identify remnant(s) of nodules Too-many small B-cells

PRIMARY MEDIASTINAL LARGE B-CELL LYMPHOMA MEDIASTINAL GRAY ZONE LYMPHOMA CLASICAL HODGKIN LYMPHOMA (NS)

3% NHL Primary Mediastinal Large B-cell Lymphoma Young adults (30-35 ys) (DLBCL > 60 ys) >> Females Mediastinal mass + supraclavear adenopathy Rare BM involvement (3%) Frequent retroperitoneal, liver, ovary and CNS (recurr.) involvement http://www.csmc.edu/ 5 yr survival: 64% (DLBCL: 46%) Derivation from asteroid B cells in the thymic medulla? CD20

Primary Mediastinal Large B-cell Lymphoma Immunophenotype Intense and homogeneous expression of B-cell antigens (CD20, CD79a, PAX5) and B-cell transciption factors (Oct2/Bob1; PU.1) CD23 variable CD30 can be expressed (83%) No immunoglobulins and HLA-I CD20 PAX5 CD23 CD30 MAL

Primary Mediastinal Large B-cell Lymphoma and Classical HL-NS Similar age and mode of presentation Young adults F>M Mediastinal mass, often bulky Involvement of thymus, supraclavicular nodes

Primary Mediastinal Large B-cell Lymphoma (PBBCL) has a gene expression signature that differs from other DLBCL and shares many elements with CHL (Rosenwald, JEM 2003) (Savage, Blood 2003)

PMBL Analogies between PMBL and CHL: Down-regulation of B-cell signaling genes HL-C Distinctive hyperexpresssion of Jak2 and adjacent genes (PDL2), correlated with 9p24 amplification Cytokine pathways, TNF (CD30) members, extracellular matrix Similar expression of IL-13Ra, NFkB, MAL, FIG1, CD58 and dependent factors (STAT-1, TRAF1, crel, MAL, FIG1), some with similar protein expression Med-LBCL NFkB

36 cases 21 MGZL 6 composite 9 sequential morphology 10 Med-LBCL 11 HL-C-NS

In Mediastinal Grey Zone Lymphomas there is an asynchrony between morphology and immunophenotype morphology 10 Med-LBCL 21 MGZL 11 HL-C-NS CD15 CD20 CD30 PAX5 BOB1/OCT2 MAL Expected and Found Reactivity Med- LBCL-like - + ++ variable +/- ++ ++ + ++/++ +/+ ++ + HL-C-NS-like + - -/+ ++ ++ + + weak + -/-, +/-, -/+ +/+ -/+ +

CD20 CD15 CD79a

CD20 Morphology suggests Med-LBCL, phenotype more like CHL CD20 +/- CD30 + CD15 ++ CD30 CD15

MGZL - Classical Hodgkin s like morphology CD20

Predominantly mediastinal tumors Clinical features similar to NS-CHL & PMBL, except for male predominance MGZL cannot be readily classified as either PMBL or CHL (asynchronous histology & immunophenotype) Challenge to pathologist and clinician B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma

Microdissected tumor cells in MGZL, PMBL, and mediastinal NS-CHL, and DLBCL Large-scale DNA methylation analysis PCA distinct epigenetic profile of MGZL intermediate between CHLNS and PMLBCL, but clearly different from that of DLBCL Class prediction models using selected genes distinguish between CHLNS, PMBL & MGZL CONCLUSION Gray zone features are present at the epigenetic level, in addition to morphology and immunophenotype Distinct entity

The optimal therapy for MGZL has not been defined and only a single study has been performed (Dunleavy, NCI 2009) Survival of 16 patients with CD20-positive MGZL treated prospectively with the DA-EPOCH-R regimen + PMBL patients with similar clinical characteristics: MGZL event-free (45 %) and overall survival (75 %) rates significantly inferior than PMBL High percentage of MGZL requires mediastinal radiation to achieve long term CR DA-EPOCH-R EFS PMBL MGZL

B-cell lymphoma, unclassifiable, with features intermediate between Diffuse large B-cell lymphoma and Burkitt lymphoma. Nightmare (Johann Heinrich Füssli, 1781)

BURKITT LYMPHOMA Typical small-non cleaved morphology CD10 + BCL6 + BCL2 - Ki67: 100% cmyc simple translocation with heavy or light chain Ig-gene Others translocations (BCL2, BCL6, CCDN1, PAX5) unaccepted! Bcl2 Ki67 Acceptable exceptions (still BL) 1. Lack of DEMONSTRABLE cmyc translocation by FISH in an otherwise typical BL especially in children and young adults (5%-10% of cases) or 2. Minor cell-size variability (Ex: atypical BL ) or myc 3. Weak and focal expression of BCL2 in an otherwise typical BL Bcl2

Morphologically a mix of cells resembling BL and larger cells resembling DLBCL, with variation of nuclear size and contour not acceptable for BL + Proliferation fraction, starry-sky pattern, prominent apoptosis and immunophenotype (CD20+/BCL2-/BCL6+/CD10+) consistent with BL or Morphologically consistent with BL, but atypical immunophenotype: strong and diffuse BCL2 positivity Ki67 < 95% or genetic features not consistent with BL (cmyc translocation with genes other than immunoglobulin genes; cmyc amplifications; complex cmyc rearrangements; BCL2 and/or BCL6 rearrangements, associated or not with cmyc)

B-cell lymphoma, unclassifiable, with features intermediate between Diffuse large B-cell lymphoma and Burkitt lymphoma. Examples BCL2 Ki67 MYC BCL2

B-cell lymphoma, unclassifiable, with features intermediate between Diffuse large B-cell lymphoma and Burkitt lymphoma. Examples Fenotipo BL-compatibile. Triple hit BCL2 BCL6 cmyc

Hummel M., et al. A Biologic Definition of Burkitt s Lymphoma from Transcriptional and Genomic Profiling. N Engl J Med 2006;354:2419-30. Dave S.S., et al. Molecular Diagnosis of Burkitt s Lymphoma. N Engl J Med 2006;354:2431-42. Burkitt Lymphomas (BL) Atypical Burkitt Lymphoma (abl) Diffuse Large B Cell Lymphoma (DLBCL) Molecular classification Molecular BL (mbl) Molecular non-bl (non-mbl) Intermediate lymphomas (mil) mbl non-mbl mil Molecular classification Histological counterpart >> BL + abl << DLBCL >> DLBCL >> DLBCL

Hummel M., et al. A Biologic Definition of Burkitt s Lymphoma from Transcriptional and Genomic Profiling. N Engl J Med 2006;354:2419-30. Dave S.S., et al. Molecular Diagnosis of Burkitt s Lymphoma. N Engl J Med 2006;354:2431-42. Molecular classification mbl Histological counterpart >> BL + abl << DLBCL Genetic anomalies non-mbl mil >> DLBCL >> DLBCL Heterogeneous Enriched of cases with IG/MYC and non IG/MYC, with complex karyotype Molecular classification + Cytogenetic Clinical >> IG/MYC simple mbl + MYC simple Favorable non-mbl / mil + MYC anomalies Unfavourable

A novel concept of high risk lymphomas that include BLU but also a significant fraction of DLBCL

DLBCL-NOS Note di terapia (Dr. G. Rossi, Ematologia, Brescia) Il sottogruppo di DLBCL nel quale la terapia attuale viene considerata del tutto insufficiente sono i linfomi con riarrangiamento di c-myc isolato o associato a altri riarrangiamenti (bcl-2, bcl-6). La sopravvivenza in questi casi è del 40% circa con terapia standard, mentre sembra che regimi terapeutici più impegnativi, in infusione continua o contenenti methotrexate ad alte dosi, come nei linfomi di Burkitt, possano dare risultati migliori. Nei pazienti anziani è dimostrato che quelli che risultano fit secondo i parametri di una valutazione geriatrica multidimensionale possono tollerare bene la terapia standard ottenendo risultati sovrapponibili a quelli dei giovani.

Kramer, Blood 1998 Macpherson, JCOl 1999 Kawasaki, Leuk&Lymph 2001 Nakamura, Mod Pathol 2002 AU, Leukemia 2004 Kusumoto, AJSP 2004 Haralambieva, AJSP 2005 Kanungo, Mod Pathol 2006 McClure, AJSP 2005 Bertrand, Leukemia 2007 Le Gouill, Haematologica 2007 Salaverria, Haematologica 2008 Yoon, Histopathology 2008 Klapper, Leukemia 2008 Mead, Blood 2008 Copie-Bergman, JCO 2009 Niitsu, Leukemia 2009 Tomita, Haematologica 2009 Stasik, Haematologica 2009 Johnson, Blood 2009 Barrans, J Clin Oncol 2010 Shustik, Haematologica 2010 Snuderl, Am J Surg Pathol 2010 Cuccuini, Blood 2012 Shaoying, Mod Pathol 2012 Kabayashi, Jpn J Clin Oncol 2012 Kanagal-S., Histopathology 2012 Pedersen, Eur J Haematol 2013 Hu, Blood 2013 Pillai, Am J Surg Pathol 2013 Horn, Blood 2013 Visco, Haematologica 2013. Studies on MYC, BCL2 and BCL6 genetic anomalies in DLBCL The occurrence of any anomaly, but especially translocations of MYC or MYC+BCL2 (double hit) pinpoints high risk DLBCL Common DLBCL IPI as prognosticator Effective treatment: R-CHOP or high doses CT if high IPI Cure: ~70% High risk DLBCL No favorable prognostic factors No effective therapy yet (variety of regimens) Cure: unlikely Median OS: 0.2-1.2 years

Review of 303 patients with de novo DLBCL, treated with R-CHOP MYC-r: 35 (14%) (74% double-hit MYC+BCL2) Survival at 2 yrs: 35% vs. 61%

Niitsu, Leukemia 2009

Series of DLBCL with >100 cases Total cases SH/DH/TH Myc+ Single Hit Double Hit Triple Hit Myc+ BCL2+ BCL6+ BCL2+ Myc+ BCL6+ Myc+ BCL2+ BCL6+ Myc+ Klapper, 2008 177 14 14 Yoon, 2008 156 14 11 5 24 1 1 1 Savage, 2009 135 12 9 3 Niitsu, 2009 394 43 24 18 19 Obermann, 2009 220 9 7 1 1 Barrans, 2010 245 35 6 55 64 19 3 7 Pedersen, 2010 157 20 3 47 17 Akyurek, 2011 239 21 14 36 69 4 2 1 Cuccuini, 2012 161 28 7 13 4 4 TOTAL 1884 196 95 161 157 77 11 13 % 100 10,40 5,04 13,52 24,53 4,51 1,08 1,62 12.25% 8.3% Brescia 160 13 7 19 17 4 1 1 % 100 8,3 4,4 11,9 10,9 2,5 0,6 0,6

Features of DLBCL with MYC ± BCL2 anomalies Clinical Mean Age: 51-65 years (exceptional < 18) Previous low-grade LNH: rare Frequent: LDH++, high stage, extranodal sites (BM, SNC, pleura) IPI prevalent: 3-4/5 Phenotypical CD10+: 88% BCL6+: 75% MUM1/IRF4+: 17%

BCL2 MIB1 MYC

MIB1 BCL2 MYC BCL6

BCL2 Myc MIB1 BC2

BCL2 MYC JH CEP8

Linfomi B diffusi, con aspetto blastoide Generalmente classificati nelle forme inclassificabile BL-DLBCL (BCL-U) o DLBCL (small centroblasts) 24 casi, de novo o secondari 9/15 casi (60%) double hit MYC-BCL2 (tutti compresi nella categoria BCL-U) 10/16 casi (63%): MYC 13/18 casi (72%): BCL2 NB: prognosi infausta in tutti i casi

Ricerca di traslocazioni multiple BCL-U DLBCL con: alto indice apoptotico-proliferativo intensa espressione di BCL2 citologia blastoide tutti?

From gene to protein

MYC BL (#1682-12) Rb-Mab Y69 (Epitomics)

MYC+ BCL2+ (immunohistochemistry)

BCL2 MYC 1. More than 2,000 patients analyzed in six studies (*) 2. MYC-BCL2 DH IIC DLBCL are significantly more frequent than DH FISH (20%-30%) of DLBCL 3. DH MYC IIC /BCL2 IIC is present in both GCB and ABC DLBCL (defined with IIC or GEP), suggesting heterogeneous molecular pathways may be responsible for MYC and BCL2 deregulation 4. The DH MYC IIC /BCL2 IIC phenotype in DLBCL is predictive of a significantly ominous outcome 5. DLBCL with MYC-BCL2 DH IIC do not respond to R-CHOP standard treatment 6. MYC-BCL2 DH IIC DLBCL occur among the elderly in the majority of cases (>65 yrs) (*) Kluk, PloS ONE 2012; Green, JCO 2012; Johnson, JCO 2012; Horn, Blood 2013; Visco, Haematologica 2013; Hu, Blood 2013

E l espressione proteica di MYC in grado di selezionare i casi meritevoli di FISH? Predittività di anomalie geniche (MYC) MYC (proteina) espressione (% delle cellule) 10 20 30 40 50 60 70 80 90 100 100% dei casi (Green, AJSP 2012) 100% dei casi (Tapia, Histopathology 2012) 100% dei casi (Kluk, Plos ONE 2012) 74% dei casi (Johnson, JCO 2012) 80% dei casi (Su, Blood 2013)

Paying tribute to the fact that only recent and as yet incomplete experience has been gained for immunohistochemical stainings with the anti-myc antibody, at the moment we strongly advise the use of a combined MYC -FISH and IHC model. a possible compromise for the moment might be to use MYC staining as a screening tool first, and then hybridize all cases with MYC protein expression >20%.

Da un concetto di borderline DLBCL-BL a una nuova possibilità di stratificare prognosticamente i DLBCL-NOS

I am confused. No wait... maybe I am not.