Clinical History. Pediatric Tumors with Involvement of the Head & Neck

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1 Pediatric Tumors with Involvement of the Head & Neck John Hicks Texas Children s Hospital Baylor College of Medicine Houston, TX NO DISCLOSURES Clinical History 10 Yr-Old Hispanic Male From Mexico with Recent Loss of Mandibular Incisors Due to Painless, Rapidly Expanding Lytic Mass Overlying Gingiva and Mucosa without Erosion or Ulceration Referred for Biopsy and Treatment 1 2 Ewing Sarcoma: Tumor-Defining ETS Translocations CD99 NKX2.2 FLI-1 EWSR1-FLI1 t(11;22)(q24;q12) 90-95% Type 1 EWS Exon 7 fused to FL1 Exon 6 (65%) Type 2 EWS Exon 7 fused to FLI1 Exon 5 (20%) 16 Other EWS-FLI1 Fusion Types (15%) EWSR1-ERG t(21;22)(q22;q12) 5-10% Type 1 EWS Exon 7 fused to ERG Exon 6 (60%) 4 Other EWS-ERG Fusion Types (40%) EWSR1-ETV1 t(7;22)(q22;q12) <1% EWSR1-EIAF (ETV4) t(17;22)(q21;q12) <1% EWSR1-FEV t(2;22)(q33;q12) <1% FUS-ERG t(21;16)(q22;p11) <1% FUS-FEV t(2;16)(q35;p11) <1% 3 4 1

2 Ewing and Ewing-Like Sarcomas Three Genetic Types EWSR1 fused to ETS-like transcription factors EWSR1 fused to non-ets-like factors Non-EWSR1 fusions Heterogeneous clinical setting and therapeutic responses Rare lesions, but common in consultation practice EWS-Non ETS Ewing-Like Sarcoma -Some Typical Ewing Features -Most Atypical Ewing Features -Osseous & Extraosseous Tumors -Variable CD99 Positivity EWSR1-NFATC2 Activates same downstream pathway as FLI1 and ERG fusions EWS-R1PATZ1 (EWSR1-ZNF278), & EWSR1-SP3 More aggressive and drug resistance similar to DSRCT EWSR1-SMARCA5 Chromatin remodeling gene fusion, similar to INI1 (SMARCB1) Suggests related to rhabdoid tumor, but not proven EWSR1-POU5F1 (OCT4) Transcription factor that regulates stem cells Heterogeneous morphology, with areas containing nested polygonal and spindle cells S100 positive Probably a form of soft tissue myoepithelial tumor 5 6 Case History 37 Week Gestation Male Neonate Delivered Vaginally At Delivery, Purple-Blue 8 cm Mass Protruded from Left Facial/Ear Region Mass Not Present on Ultrasound at 20 Weeks Gestation Diagnostic Imaging and Clinical Impression: Congenital Infantile Hemangioma (Vascular Tumor) Prednisone and Lansoprazole Therapy Initiated Tumor Rapidly Increased in Size & Ulcerated Resection Performed on 8th Day of Life 7 8 2

3 Flow Cytometry Results CD56 (NCAM) CD38 Positivity Interpretation: Not Compatible with Hematopoietic/Lymphoid Neoplasm FISH for MYC-N No Evidence of Amplification 9 Routine Cytogenetics Pending 10 Immunohistochemistry: Negative Myogenic Desmin, Myogenin, MyoD1 Epithelial EMA, Pancytokeratin, CAM 5.2 Lymphoid LCA, CD20, CD3, TdT, CD61, CD31, Myeloperoxidase, CD30, CD43, ALK1, CD4, CD8, CD68, CD1a Vascular CD34, CD31 Neural *PHOX2B, NB84, S100, NFP, NSE, Chromogranin Germ Cell PLAP, CD30, AFP Melanocytic HMB45, S100 Protein

4 13 14 Cytogenetics FISH Breakapart Rearrangement of EWSR1 Splitting and Rearrangement of 5 EWSR1 to 20q11.2 (FISH requested after EM Findings) Conventional Cytogenetics 50,XY,+6,+12,+15,+17,t(20;22)(q11.2;q12) 51,idem,+der(22)(t20;22)(q11.2;q12) Diagnosis Peripheral Primitive Neuroectodermal Tumor (Ewing Sarcoma) with Probable Novel t(20;22) EWSR1-NFATC2 Translocation

5 Ewing Sarcoma/pPNET: 15% Before Age 5 Yrs 22 Congenital Ewing Sarcoma Cases 40% Metastatic Disease (Skin, LN, Brain) DOD (15 of 22) At 1-24 Months 17 Re-Assessment Several Years Later By NGS MN1-ZNF341 Translocation MN1 (22q12.1) and ZNF341 (20q11.22) EWSR1 (22q12.2) and NFATC2 (20q12) MN1: Transcriptional activator regulates expression of TBX22 in palate development and fusion of the palatal shelves Promotes maturation and normal function of calvarial osteoblasts, including osteoclastogenic cytokine TNFSF11/RANKL expression. Necessary for development of the membranous skull bones May play a role in tumor suppression. ZNF341: Nuclear Zinc Finger Transcription Ractor Regulates Expression of STAT3 - A Central Regulator of Immune Homeostasis 18 SRCT: Atypical Ewing-Like Sarcoma Ewing-Like Sarcoma 90 to 95% 5 to 10% BCOR-CCNB3 MN1-ZNF341 WT1 Calretinin CD

6 CIC-DUX4 SRCT CIC-DUX4 SRCT: Clinical Features Most Common Fusion in EWSR1-Negative SRCTs (up to two-thirds of cases) >1 DUX4 Copy in Some Tumors Due to Duplication Events t(4;19)(q35;q13) and t(4;10)q26.3;q13) Defective Splicing of DUX4 Muscular Dystrophy 21 Male Gender (4M:1 F) Median Age: 24 Years (range 6-62 years) Extremity Tumors: 50% Metastatic Disease: 50% CD99: Most Diffusely Positive, Patchy in Some WT1 & Calretinin Positive FLI: Variable Rare: EMA, Cytokeratin, S100 and Desmin Negative: Chromogranin, Synaptophysin AE/AE3 WT1 Calretinin CD99 NKX2.2 MUC4 22 CIC-DUX4 Fusion Protein: CIC Binding Site for TLE proteins, similar to Synovial Sarcoma DNA binding site (DUX4) Upregulates several ETS family genes, similar to Ewing Sarcoma type fusions Alternate partners for CIC described, similar to EWS CIC-DUX4, CIC-DUX4LI0, CIC-FOXO4 SRCT: Atypical Ewing-Like Sarcoma

7 Immunostain Profile BCOR3-CCNB3 SRCT: IHC CCNB3 nuclear 100% BCOR 100% CD99 membranous 16% CD99 cytoplasmic 24% CD99 dot-like (Golgi) 48% Desmin 0% EMA 0% Pancytokeratin 0% S100 protein 0% SMA 0% CD34 0% CCNB Usually Arises in Bone, Also Occurs in Soft Tissues Rare Tumor <5% Fuses BCOR, an Epigenetic Repressor, to Cyclin B3 Amplifies Ability of Cyclin B3 to Drive Cell Cycle Events Causes Loss of Function of BCOR, Resulting in Epigenetic Instability BCOR-CCNB3 SRCT BCOR Gene Transcriptional Repressor Associated with BCL6 (Xp11.4) Oncoprotein and Histone Modifying Enzymes Acts as Suppressor of Gene Expression Through Epigenetic Mechanisms. Regulation of Mesenchymal Stem Cell Function in Early Embryogenesis Contributes to Laterality Determination 27 BCOR3-CCNB3: Outcome Median age: 13.1 yrs (range 5.9 to 25.6 yrs) Gender Ratio: 2 Male: 1 Female Age Groups: 1-9 years 23% years 50% >18 years 27% 5 Year Overall Survival 80% 5 Year Disease Free Survival 71% Histology Type Small Round Cell 51% Spindled 100% Mixed 88% Relapse 36% Local 37% Metastatic 13% Both 50% 28 7

8 Clinical History SRCT: Atypical Ewing-Like Sarcoma 2 year old female presents with a 1 year history of right jaw pain. Father first noticed it when he touched her jaw to kiss her and she cried. Pain was intermittent but has become more frequent. Increasing difficulty opening her mouth due to pain and her teeth have come out of alignment. Patient has been gaining weight, but now will only eat soft food. No facial weakness or paresthesias reported INI-1 Cyclin D1 BCOR WT-1 NGFR Vimentin

9 What Was Expected: Clear Cell Sarcoma of Kidney BCOR EXON 15 PCR (ITD) Cytogenetics: normal male karyotype. Whole exome & RNA sequencing: -BCOR Internal Tandem Duplication Undifferentiated Round Cell Sarcoma of Infancy & Primitive Myxoid Mesencyhmal Tumor of Infancy with BCOR Internal Tandem Duplication AJSP August 2016 (40: BCOR Gene BCOR Mutations: Oculofaciocardiodental and Lenz micro-ophthalmia syndromes; myeloid leukemia; myelodysplastic syndromes; medulloblastoma BCOR-RARA t(x;17): acute promyelocytic leukemia BCOR-ZC3H7B t(x;22): Endometrial stromal sarcoma and Ossifying fibromyxoid tumor BCOR ins(4;x): Ewing-like undifferentiated round cell sarcoma BCOR-MAML3 & ZC3H7B-BCOR: Undifferentiated Small Round Cell Tumors

10 BCOR Gene BCOR-HGNET: CNS-PNETs (medulloblastomas) BCOR-CCNB3: Undifferentiated Round Cell Sarcoma (CCNB3 IHC) NKX2.2 Positive NKX2.2 Negative /Fusion Panel BCOR ITD: Clear Cell Sarcoma of Kidney Reported in 15/29 (52%) Infantile Sarcomas (9/22 Infantile Undifferentiated Round Cell Sarcomas and 6/7 Primitive Myxoid Mesenchymal Tumors of Infancy) / Fusion Panel NGS MN1/ZNF CIC-DUX4LI0, CIC-FOXO4, BCOR-iTD 38 Rhabdomyosarcoma Alveolar RMS Most Common Malignant Soft Tissue Tumor (60% of All Sarcomas) in Children 4.7 per 1,000,000 Children Derived from Primitive Mesenchyme Embryonal RMS: Infants & Young Children Botryoid ERMS: Mucosal Surfaces Spindle Cell RMS: Paratesticular, Most Common Alveolar RMS: Adolescents, Extremities Sclerosing RMS: Most in Extremities Anaplastic RMS: More Aggressive Behavior Pleomorphic RMS: Older Adults

11 Desmin Typical PAX-FOXO1 Rearrangements: Alveolar Rhabdomyosarcoma PAX3-FOXO1 PAX7-FOXO Spindle Cell & Botryoid RMS Embryonal RMS and Strap Cells

12 Desmin RMS: IHC Myogenin (nuclear) MyoD1 (nuclear) MSA & SMA PAX5 (Alveolar RMS) Creatine Kinase M Vimentin (most primitive) Keratin Neuroendocrine Markers p53 45 Alveolar RMS: PAX3/FOXO1 (most) PAX7/FOXO1 (next most) PAX3/FOXO4, PAX3/NCOA1, PAX3/NCOA2 FOXO1/FGFR1 CDK4 Amplification Mutations: TP53, CDKN2A, CDKN2B, FGFR4 ALK Copy Gain Tumor Suppressors: RASSF, HIC1, CASP8 DNA Methylation Genetics: RMS Embryonal RMS: 11p15.5: IGF2, H19, CDKN1C, HOTS Mutations: RB, TP53, CDKN2A, CDKN2B, RAS, FGFR4, PIK3CA, CTNNB1 (beta-catenin), MyoD1 NF1 Deletions ALK Copy Gain DNA Methylation 12q13: GLI 46 5-Year Event Free and Overall Survival by Molecular RMS Subtype Event-Free Overall Survival Survival Embryonal RMS 77% 82% Alveolar RMS PAX3-FOXO1 54% 64% PAX7-FOXO1 65% 87% *Alveolar Pattern, BUT Fusion Negative 90% 89% 47 Fusion-Negative RMS: Survival MG5: Molecular Gene Score Based on 5 Genes EPHA2 - Ephrin Receptor A2 EED - Embryonic Ectoderm Development NSMF - NMDA Receptor Synaptonuclear Signaling and Neuronal Migration Factor CBS - Cystathionine-b-Synthase EPB41L4B - Erythrocyte Membrane Protein Band 4.1 Like 4B 48 12

13 MyoD1 Mutation in RMS Infant with Maxillary Tumor with Spindle Cell Features Myogenin Desmin MyoD Fluorescent In Situ Hybridization Findings DESMIN Myogenin \ MyoD1 Initial H&E Impression: Rule Out Infantile Fibrosarcoma ETV6 and NTRK3 FISH Negative

14 Cytogenetic & Molecular Findings TEAD1-NCOA2 Rearrangement Recently Described with Infantile Spindle Cell Rhabdomyosarcoma FOXO1 Rearrangement Negative 53 4 wk old child with NCOA2-TEAD1 Fusion 7 mo old male with NCOA2-SRF Fusion 3 mo old child with NCOA2 Fusion Distinctive Herring Bone-Like Fascicles Reminiscent of Fibrosarcoma with High Grade, Hypercellular Components with High Mitotic Activity and Scattered Pleomorphism No Rhabdomyoblasts on Routine H&E Staining Mimics Infantile Fibrosarcoma 54 Fusions in Infantile Spindle Cell RMS NCOA2 at 8q13 Fusion With: SRF at 6p21 TEAD1 at 11p15.2 VGLL2 at 6q22 VGLL2-CITED Inversion: VGLL2 at 6q22 CITED at 6q NCOA2 of VGLL2 Infantile RMS Follow-Up No Metastatic Disease in All Reported Patients One Patient with Local Recurrences at 1 & 3yrs - But Disease Free at 15yrs Another Patient with Local Recurrence at 10mos, But Disease Free at 3yrs Alive with No Evidence of Disease at Follow-Up (median 7yrs, range 3 to 15yrs) 56 14

15 VGLL2 Or NCOA2 Rearranged Tumors NO VGLL2/NCOA2 Rearranged Infantile Spindle Cell RMS Patients Developed Metastatic Disease Raises Question: Should These Tumors Be Considered High- Grade Tumors Similar To other RMS Subtypes? Lack of Metastatic Potential May Advocate Against Need for Systemic Chemotherapy if Adequately Excised Molecular Findings in Infantile Spindle Cell and Sclerosing Rhabdomyosarcoma Congenital Spindle Cell (infantile) and Sclerosing RMS (adult ) VGLL2-CITED2 VGLL2-NC0A2 TEAD1-NCOA2 PAX3-NCOA2 SRF-NCOA2 MyoD1 Mutations VGLL2 and NCOA2 Fusions and Prognosis in Rhabdomyosarcoma Congenital Spindle Cell and Sclerosing Rhabdomyosarcoma VGLL2-CITED2 VGLL2-NC0A2 TEAD1-NCOA2 PAX3-NCOA2 SRF-NCOA2 Less Aggressive & Favorable Outcome Mutations and Prognosis in Rhabdomyosarcoma Pediatric Spindle Cell (greater than 1 year of age) and Sclerosing RMS: MyoD1 Mutations PIK3CA Mutations MyoD1 and PIK3CA Mutations Aggressive Tumors Unfavorable Outcome