3/31/2017. Disclosure of Relevant Financial Relationships

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1 Disclosure of Relevant Financial Relationships USCAP requires that all planners (Education Committee) in a position to influence or control the content of CME disclose any relevant financial relationship WITH COMMERCIAL INTERESTS which they or their spouse/partner have, or have had, within the past 12 months, which relates to the content of this educational activity and creates a conflict of interest Identification LYMPHOBLAST 1958 Identification LYMPHOBLAST Morphology & Immunophenotyping T ASSOCIATED PLASMA CELL PLASMACYTOID T CELL PLASMACYTOID MONOCYTE Identification LYMPHOBLAST 1990 Morphology & Immunophenotyping T ASSOCIATED PLASMA CELL PLASMACYTOID T CELL PLASMACYTOID MONOCYTE Cell function PLASMACYTOID TYPE I INTERFERON PRODUCING CELL Biological features in vivo & in vitro Role in human diseases neoplasms

2 NEG POS phenotype (2017) Cell Lineage Negative (lin negative ) BDCA2 (CD303) E2 2 CD19, CD20, CD79a, PAX5, sig, cig CD2AP BCL11a SPIB BCL7A BAD LAMP TdT CD34 CD117 Myeloperoxidase, CD11c, CD14, CD13, CD33, CD163 Lysozyme, ASD CAE, esterases CD3, CD5, CD8, LAT, ZAP70, TCRAB, TCRGD Perforin, TIA1 CD16, CD4 CD43 CLA CD36 CD68 Granz B : high expression on Most widely used marker Other normal cells are positive BDCA2/CD303 clone 124B3.13 HEV Epitheliod macrophages Sinus lining cells Ma J. Biophys Rep 2015; 1: Ceribelli M, Cancer Cell 2016;30: Antibodies recognizing the master transcription factor E2-2 (TCF-4) on in paraffin sections E2-2 / TCF4 (Rabbit-monoclonal anti-human TCF4, clone 6A by Epitomics) (Antibody courtesy by Dr. Ceribelli M., Louis Staudt Lab, NIH) E2-2/TCF4 BDCA2 2

3 Abundant Superficial LNs Tonsils and adenoids Young individuals Rare Deep LNs Eldery individuals Thymus (medulla) Spleen (MZ area) Bone marrow Gut Occurrence in Normal Tissues % % of PBL Marked increase of in reactive processes Lymph Nodes Kikuchi s lymphadenitis Castleman (HV) disease Skin Lupus erythematosous Kikuchi s lymphadenitis HV Castleman s disease MZ Almost absent in non-lymphoid (human) tissues Lupus E. IRF7 J Exp Med 1997; 185: Costitutive expression of: A. IRF7 master regulator of INF-type I transcription B. Myxovirus resistance protein 1 (MxA) key mediator of the INF-type I-induced antiviral response MxA Cell function PLASMACYTOID TYPE I INTERFERON PRODUCING CELL Cell function PLASMACYTOID TYPE I INTERFERON PRODUCING CELL Anti-IFN- Hematopoietic Dendritic Cells MAIN CATEGORIES Classical (Myeloid) DC CD1c+ (CD141 ) cdc (CD1c+) CD141+ cdc Plasmacytoid DC (+ CD303+) Langerhans Cells (Langerin/CD207+) Swiecki M, Colonna M,. Nat Rev Immunol 2015; 15: 471 3

4 HSC Onai N, et al. Nat Immunol 2007 Auffray C, et al. JEM 2009 Liu K, et al. Science 2009 Lee J, et al. JEM 2015 Breton G, et al. JEM 2015 HSC MYELOID PROGENITORS GRANULOCYTE-MONOCYTE PROGENITOR MONOCYTE COMMON CIRCULATING MP GMP MDCP CDCP DCP PMN Monoc pdc + CD303+ cdc CD1c+ cdc CD141+ Da Geissman F. (JEM, 2015) The real thing: How to make human DC subsets Plasmacytoid DC Classical/Myeloid DC (CD1c+) Classical/Myeloid DC (CD141+) MYELOID PROGENITORS GRANULOCYTE-MONOCYTE PROGENITOR MONOCYTE COMMON CIRCULATING MP GMP MDCP DCP 1 PMN Monoc 2 2 CDCP 3 pdc + IRF4 Batf3 CD303+ cdc CD1c+ cdc CD IRF8and GATA2 involved in GMP to MDCP transition (GATA2 influences also B and NK development) FLT3L (CD135) promotes generation of DCs from progenitors TCF 4 (E2 2) 3 SpiB, BCL11a, IRF8, RUNX2 (TCF 4 targets) development & differentiation ( identity) NEOPLASMS derived from Tumoral proliferations of Mature Not recognized as a distinct entity by WHO Immature Blastic Plasmacytoid Dendritic Cell Neoplasm (WHO, 2008 & 2016) Agranular CD4+ natural killer cell leukemia (Brody JP; 1995) Blastic natural killer leukaemia/lymphoma (DiGiuseppe JA; 1997) Blastic NK-cell lymphoma (WHO; 2001) Agranular CD4++ haematodermic neoplasm / tumor (Petrella; 2002) / Herling; 2007) Mature Blastic Neoplasm Basic disease Myeloid neoplasm (mostly CMML) proliferation 10% 20% associated with myeloid neoplasm (MDS, AMoL) Age/Sex Median: 69 (6 88) M/F = 7/1 Evolution Evolution of the associated myeloid neoplasm ( may regress) Vitte s series (2013): 16 cases 52.4% alive Mean from skin onset: 23 months 45.2% dead of disease (8.75 months) Median: 65 (0 96) (5% in <10 years old) M/F = 3/1 Systemic dissemination common Median survival: ~ 14 months (9 20) NEOPLASMS derived from NEOPLASMS derived from Most common sites of diagnosis Histopathology Mature Bone marrow, Lymph node, Skin Biopsies perfomed during staging (BM) or for evaluation of lymphadenopathy or skin eruption Nodules or aggregates of cells cytologically similar to normal No/few mitoses Apoptotic bodies frequent In skin biopsies abundant inflammatory component (macrophages, lymphocytes, indeterminate DC) Leukemic cells may be also present in the same biopsy Blastic Neoplasm Skin, Bone marrow, Lymph node, Peripheral blood Immature blasts Frequent mitoses No apoptosis No/minimal inflammatory component Mature None Occasional CD2, CD5, CD7, CD10, CD13, CD14, CD15, CD33, weak TdT: negative Ki 67 <10% Aberrant expression of markers usually positive in normal Aberrant expression of markers usually negative in normal Blastic Neoplasm CD68 and CLA (negative or positive as paranuclear dots) Granzyme B: negative Consistent Frequent CD7, CD33 Occasional CD2, CD5, CD7, CD10, CD13, CD117, MUM1, BCL6, S100 TdT: positive (30%) Ki 67 >30% 4

5 NEOPLASMS derived from mature NEOPLASMS derived from mature CLA 24, M (CMML) Lymphadenopathy Skin papular eruption AML (DOD, 8 months) MPX (CMML) CD68R BDCA2/CD303 NEOPLASMS derived from mature CD34 AML M; 48 AML (MO/M1) CD68R NEOPLASMS derived from Mature None As in normal is negative Aberrant expression of markers usually positive in normal Aberrant expression of markers usually negative in normal Blastic Neoplasm CD68 and CLA (negative or positive as paranuclear dots) Granzyme B: negative Consistent Frequent CD7, CD33 Cluster of Reactive LN Occasional CD2, CD5, CD7, CD10, CD13, CD14, CD15, CD33, weak TdT: negative Ki 67 <10% Occasional CD2, CD5, CD7, CD10, CD13, CD117, MUM1, BCL6, S100 TdT: positive (30%) Ki 67 >30% 5

6 tumor in CMML Lymph Node Reactive LN Clonal relationship between the myeloid neoplasia and the tumor has been proven in several cases by showing the same (not recurrent) genetic anomaly in the two cell components (FISH) or mutation (NGS)* Mongkonsritragoon W, Am J Clin Pathol 1998 Vermi W, Am J Surg Pathol 2004 Chen YC, Am J Clin Pathol 2003 Pileri SA, Leukemia 2007 Dargent JL, J Cutan Pathol 2016 * Bodmer A, Virchows Arch 2017 (MDS And : non-germline mutation of PTPN11 (c.1502g>a) 61, M CMML Erythematous skin rash BM aspirate: trisomy 13 Blastic Plasmacytoid Dendritic Cell Neoplasm (WHO, 2008 & 2016) BN is a clinically aggressive tumour derived from the precursors of plasmacytoid dendritic cells (also known as professional type 1 interferon producing cells or plasmacytoid monocytes), with a high frequency of cutaneous and bone marrow (BM) involvement and leukaemic dissemination. BDCA2 Presentation Rare (exact incidence unknown) (665 cases as series or single reports ) No racial or ethnic predominance No etiology known (EBV, HHV8, and other viruses negative) SKIN LEUKEMIA M/F: 3/1 Median age at diagnosis: 65.0 years (M: 67 y; F: 58 y) Age peak only for males 5% in 10 years Asymptomatic cutaneous lesions Good general health (lasting even months) Interval between first symptoms and diagnosis: 1 18 months (mean: ) Not recognizable other clinical manifestation in 40%-50% of cases Systemic dissemination invariably (often rapidly) occurs Elevated WBC count, circulating blasts, massive bone marrow infiltration Leukemia without skin lesions in ~7% of cases 6

7 SKIN Any site, any size (few mm. several cm.) Variable appearance (Courtesy: L. Cerroni) (Courtesy: T. Petrella) Presentation SKIN OTHER SITES AT ONSET Bone marrow: 50%-90% Can be minimal and demonstrable only by immunohistochemistry Increases with progression Lymphadenopathy: 40% Local or disseminated Splenomegaly: 25% Peripheral blood: Counts generally low (median 2%) Increase with progression. CNS Rare at presentation; frequent on relapse 71, M ( ) 7

8 Low density may simulate inflammation DIAGNOSIS Leukemia cutis (AML, ALL)? BN? EXCLUSION IRRELEVANT INCLUSION CD3, CD20 MPO, CD11c, CD14, CD163 Lysozyme CD34 CD2, CD7, CD79a CD33 TdT, CD117 S100 CD4 BDCA2/CD303 CD2AP BCL11a MXA 8

9 CD4+ + coexpressed in >90% of cases Despite their original defining role (CD4+ + Hematodermic Neoplasm), they should not be used as the only markers for BN diagnosis Other hematological malignancies CD4/ positive (esp: AML/AmoL) LYS CD4 CD4 Acute Monocytic Leukemia 237 cases of BN (all 4 markers applied) CD % CD % CD % : 95% AML: generally negative/deam on IHC, but frequently expressed on flow cytometry : 89% AML: negative; ALL: frequently positive BDCA2/CD303: 79% CD4-1 BDCA2/CD303 Neg Pos BN (n=21) 2 19 AML (all FAB subtypes) (n=55) 55 0 ALL(15 B ALL; 5 T ALL) 15 0 DLBCL (n=35) 35 0 PTCL (n=20) 25 0 Garnache-Ottou F, et al. Br J Haematol 2009;145:624 BDCA2 represents the most specific marker for leukemia using flow-cytometry Immunophenotypic criteria for BN diagnosis especially to differentiate from AML Practical approach Homogeneous and strong E2-2/TCF4 expression in: BN cases (24/28) BN or CMML with pdc proliferation (2/2) Unclassified PHN 3/13 AML (0/10) Positivity for at least 3 of 5 among CD4 BDCA2 + Negativity for CD3 CD20 MPO Lysozyme Cronin DMP, Am J Clin Pathol 2012 Sangle NA, Mod Pathol 2014 Julia F, Am J Surg Pathol 2014 Facchetti F, Mod Pathol

10 MOLECULAR & GENETICS GENE EXPRESSION PROFILE STUDIES COMPARED TO NORMAL CELLS Closer to normal than to myeloid and lymphoid precursors (Sapienza, 2014) Compared to normal Upregulation of, CCND1, IRF4 NFKB pathway activation (Sapienza 2014) MyP LyP MyP BN MOLECULAR & GENETICS GENE EXPRESSION PROFILE STUDIES COMPARED TO OTHER LEUKEMIAS Distinct from CMML (cutaneous), AML, ALL (Dijkman, 2007; Sapienza 2014; Ciribelli 2016) Unsup Hier Clust of top 2500 genes BN has marked overlap with for TCF-4 dependent genes, but some relevant differences exist: upregulation of, MYC, downregulation of BCL11a, SpiB, IL3RA, CLEC4C In BN TCF-4 dependent transciption is attenuated for specific functions and increased for oncogenic gene expression (Ciribelli, 2016) Sapienza, 2014 (Dijkman, 2007) (Sapienza 2014) (Ciribelli 2016) MOLECULAR & GENETICS MOLECULAR & GENETICS 66% of BN have an abnormal karyotype, with complex abnormalities in the same cells (average of 6 to 8) Gross genomic imbalances (mostly deletions and losses). Most frequent chromosomal targets: REF Gene % Jardin Alayed Menezes Menezes TET IKZF3, ZEB Tumor suppressor From onset of disease Common in many myeloid neoplasms Transcription Newly identified genes in leukemia 4 (4q34) 5q (5q21 or 5q34)(72%) 9 (losses)(9p21.3) 12p (12p13)(64%) 13q (13q13-21)(64%) 6q (6q23-qter)(50%) 15 (partial losses) 17 (partial losses) Combinations of deletions of TS genes (and related proteins): RB1 (Rb-1) CDKN1A (p27); CDKN2A (p16 ink4a, p14 arf ) TP53 (p53) G1/S dysregulation Stenzinger IKAROs family ASXL1 NRAS NPM1 NRAS ATM MET, KRAS, IDH2, KIT (each) No common affected genes between patients Half cases had mutations affecting either the DNA methylation or chromatin remodeling pathways NRAS, KRAS and ATM mutually exclusive (clinical subtypes?) Leroux D, et al. Blood 2002; 99:4154. Dijkman R, et al. Blood 2007; 109: Jardin F, et al. Leukemia 2009; 23:687. Wiesner T, et al. J Invest Dermatol 2010; 130: Lucioni M, et al. Blood 2011: 118: Jardin, Br J Haematol 2011 (15 cases - PCR+Sequencing) Alayed, Am J Hematol 2013 (5 cases -Targeted NGS (28 genes) Menezes, Leukemia 2014 (25 cases -Targeted NGS (32 genes) Stenzinger, Oncotarget2014 (33 cases - TargetedNGS (50 genes) EVOLUTION Good initial response to RT and/or CT (75%) generally followed by relapses (median 11 months) skin (100%) other sites (44%) systemic with leukemia (39%) Median survival: ~ 14 months ( ) TREATMENT At the present time no specific, effective and durable treatment available Allo-BMT (Auto-BMT) remains the most efficient approach Best results if BMT in first remission Blood 2013;121: Exceptional cases (especially with single isolated skin lesion) show long survival Dalle S. et al. BJD (2009) ALL-type induction most efficient than AML-type induction Dalle S, Br J Dermatol 2009 Pagano L, Haematologica 2013 Ross Weil D, Blood 2013 (*) Aoki T, Blood 2015 (*) Patients allografted in CR1 Patients allografted in more advanced status 10

11 SL-401. Immunotoxin: human recombinant anti-il-3r alpha () joined to diphtheria toxin overexpressed in BN No amplification nor translocation Cancer Discovery 2016 Cancer Discovery 2016 % priming % alive on VTCX CAL-1 (BN) high priming on BIM stimulation and high sensitive to VTCX 80, M BN since 18 mths Previous unsuccessful treatments Venetoclax: Optimal Skin and LN response No BM response DOD after 6 weeks 73, M BN since 15 mths Previous unsuccessful treatments Venetoclax: Optimal Skin and LN response Partial BM response DOD after 12 weeks TCF4 (E2-2) master regulator of the BN oncogenic program TCF4 downregulation causes the loss of the BN-specific gene expression program and tumor cell apoptosis TCF4 is a lineage-survival oncogene in BN The TCF4 transcriptional network is inhibited by Bromodomain and extra-terminal domain inhibitors (BETis) BETis are highly toxic to BNs, both in vitro and in vivo (xeno) BET inhibitors promising in BN treatment as single agent silencing multiple driver-genes The Floating Piers Christo and Jean-Claude Iseo Lake, Brescia, Italy 11