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Tumor Syndromes Disclosure of Relevant Financial Relationships Predisposing to USCAP requires that all planners (Education Committee) in a position to Osteosarcoma influence or control the content of CME disclose any relevant financial relationship WITH COMMERCIAL INTERESTS which they or their Meera Hameed MD 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. Dr. Meera Hameed declares she has no conflict(s) of interest to disclose.

Osteosarcoma 19 year old male water polo- athlete presented with shooting pain of right leg • Most cases are sporadic • Very small percentage occur in association with Cancer Predisposition Syndromes • Li-Fraumeni Syndrome • Hereditary Retinoblastoma • Rothmund-Thomson Syndrome (RTS Type 2) • Werner Syndrome • Bloom Syndrome • RAPADILINO Syndrome • Diamond Blackfan Anemia • Metastases to spine, right ischium and lungs • Alk Phos: >8355; LDH: 512

Osteosarcoma IMPACT: Integrated Mutation Profiling of Actionable Cancer Targets Adapted from Wagle, Berger et al., Cancer Discovery, 2:82‐93, 2012 Prepare 24‐48 libraries

Hybridize and select Sequence to 500‐1000X (HiSeq Align to genome (NimbleGen SeqCap) 2500) and analyze

Probes for 410

B B B B Sample B Bar coded Pooled lib & Base call & Call mut & B DNA sequence Lib prep capture align indels prep

Somatic Mutations (Tumor-Normal Pairs): Base Substitutions Small Indels Copy Number Alterations Select Rearrangements

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Tumor-Normal sequencing

Somatic variant Germline variant

Tumor Tumor Tumor c.4540C>T (p.R1514*)

Normal Normal Normal

G>T C>T

MSK IMPACT Results Copy Number Alterations Copy Number Fold CDK4 Alteration CDK4 (12q14.1) Amp 13.5 MDM2 (12q15) Amp 2.4 CCND3 (6p21.1) Amp 2.5 VEGFA (6p21.1) Amp 2.4 1p36 6p21 MTOR (1p36.22) Amp 2.3 PIK3CD (1p36.22) Amp 2.3 SPEN (1p36.13) Amp 2.3 SDHB (1p36.13) Amp 2.3 FAT1 (4q35.2) Del -2

ATRX exon 15 c.4540C>T (p.R1514*) 4q35

Adapted from Chen et al. Cell Rep. 2014 April 10; 7(1): 104–112.

SNP array- Copy Neutral LOH (17p13) Patient’s father with history of melanoma developed left groin mass Leiomyosarcoma

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Pedigree Li-Fraumeni Syndrome (Tumor spectrum-SBLA) 67 Lung 70 65 Lung 65 Lung Chompret Criteria Classic Criteria (2009)

62 81 81 Melanoma Proband with sarcoma Proband with LFS tumors before 46 before 45 and one or two 1st degree relatives 1st degree relative with with LFS tumors before 56 or with Melanoma & 58 60 sarcoma before 45 multiple tumors 34 30 56 59 Sarcoma 1st or 2nd degree relative or with any cancer before 45 Proband with multiple tumors of LFS or sarcoma before 60 spectrum and first one occurring

19 24 22 25 27 21 before 46 years or Osteosarcoma Patients with adrenocortical ca or ? Li-Fraumeni Syndrome choroid plexus tumors

Germline MSK-IMPACT - Secondary Secondary Germline IMPACT germline Testing Tier 1Tier 2 .Tumor and Normal Pairs APC MSH6 SDHA TSC2 ALK EGFR MET RAD50 SUFU .Option for germline analysis for 76 hereditary BAP1 MUTYH SDHB VHL ATM EPCAM MITF RAD51 TERT BRCA1 NF1 SDHC WT BARD1 FAM175A MRE11A RAD51B TMEM127

cancer predisposition genes BRCA2 NF2 SDHD BLM GATA2 NBN RAD51C .Variants –reviewed and classified CDH1 PALB2 SMAD3 BMPR1A GREM1 NRAS RAD51D FH PMS2 STK11 BRIP1 HRAS PAX5 RECQL4 .Likely pathogenic and pathogenic variants are FLCN PTEN TGFBR1 CDK4 JAK2 PDGFRA RUNX1 sent to clinical report. MEN1 RB1 TGFBR2 CDKN2A KIT PHOX2B SMAD4 MLH1 RET TP53 CHEK2 KRAS POLE SMARCA4 MSH2 SDHAF2 TSC1 DICER1 MAX PTCH1 SMARCB1

Li-Fraumeni Syndrome

 Germline mutations in TP53 Autosomal Dominant Tumor c.916C>T(p.Arg306*)  Sarcomas, Breast Carcinoma, Brain tumors Leukemia, Lung Cancer and Adrenocortical Carcinoma (SBLA)  Classic Criteria and Chompret Criteria (2009) Normal c.916C>T(p.Arg306*) Genetic anticipation

Li-Fraumeni Syndrome

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TTP53 mutations in Li-Fraumeni Syndrome Retinoblastoma Transactivation DNA binding TD RD Bilateral Leukocoria RB1

Proline Rich Distributions of mutations 175 248 273 282 245 249 306

DBD Codon 273- Rhabdomyosarcoma Adapted from: Chapter 21. Retinoblastoma Codon 245, 282- Osteosarcoma and Simulating Lesions Ralph C.Eagle JR Outside DBD Ognjanovic S etal. Sarcomas in TP53 germline mutation carriers. A review of Leiomyosarcoma the IARC TP53 database. Cancer 2012;March 1:1387-1396

Hereditary Retinoblastoma Retinoblastoma Germline Mutation Spectrum 1 2 3 7 17 RB1 18 27 1% 5’ 3’

WT/mut WT/WT 5%

7% Nonsense WT/mut 9% 37% 37% Splice Site Frame shift Large indel Zygote 20% chromosomal deletion Missense Promoter WT mut

21%

Dommering et al. RB1 mutation in a comprehensive nationwide cohort of retinoblastoma patients. J Med Genet 51:366-374, 2014.

Retinoblastoma Washington Post July 12, 2016 Second Cancers among Retinoblastoma Alfred G. Knudson Jr., geneticist who decoded a mystery of cancer, dies at 93 Survivors • Osteosarcoma “The Two-Hit Hypothesis” • Soft Tissue Sarcoma • Melanoma • Lung Cancer • Lymphoma • Bladder Cancer • Uterine Cancer Interviewed for the book “The Emperor of All Maladies: A Biography of Cancer” by Siddhartha • Breast Cancer Mukherjee, Dr. Knudson reflected on the knowledge of the disease that he divined. It • Brain Tumors Knudson AG Jr. Mutation and Cancer: Statistical study of was, he said, like inferring “the wind from the • Cancers of Mouth or Nose retinoblastoma. Proc Natl Acad Sci 68(4):820-3, 1971 movement of the trees.”

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14 year old male with bilateral retinoblastoma at 23 10 years later patient presented with right leg pain months of age s/p chemoradiation and enucleation, developed metastases to right distal femur at 4 years of age

SYN

Therapy: Radiation

Chondroblastic Osteosarcoma Bone Sarcoma in Retinoblastoma Survivors

•Standardized incidence and mortality of bone sarcoma- several hundred fold higher •Cumulative incidence is 7% over 20 years •Non-irradiated tumors- lower risk •Osteosarcoma is the most common

Kleinerman et al. Clinical Sarcoma Research 2:15, 2012

Rothman-Thomson Syndrome Retinoblastoma • Poikiloderma • Alopecia “Two-hit” Hypothesis by Dr. Alfred Knudson • Aplasia of thumb Bialleic mutation of RB1 • Valgus deformity Autosomal Dominant • Short stature Increased risk of subsequent malignancies • Frontal bossing Alfred Knudson (1922-2016) Post Radiation Osteosarcoma Nature.com • Skeletal abnormalities Mutations distributed throughout the gene family gene mutations identified in 1999

Adapted from Larissa et al. Rothmund-Thomson Syndrome Orphanet Journal of Rare Diseases 2010 (5);10:1150-1172.

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Rothmund-Thomson Syndrome RecQL4

N C A NTS1 NTS2 Helicase C G T AC  Enzymes which unwind Domain   21   1     double-helix of DNA         Preparation for cell division

G T C A  Repair of damaged DNA Helicase region T G  RecQ helicases- Cancer Truncating Mutations Frame shift Missense mutations Predispostion Nonsense Splicing Intronic deletions L. Wang et al. JNCI J Natl Cancer Inst 2003;95:669-674

Rothmund-Thomson Syndrome Rothmund-Thomson Syndrome

 RecQ Helicase associated Autosomal Recessive Disorder  RTS Type I and RTS Type II  RecQL4 helicase mutation predisposes to Osteosarcoma (RTS Type II)  Range of mutations (nonsense, frame-shift, intronic deletions)  RecQL4 mutations are not seen or only very rarely seen in sporadic osteosarcoma

Wang LL et al. Associations between osteosarcoma and deleterious mutations in RECQL4 gene in Rothmund-Thomson Syndrome. J Natl Can Inst 2003; 95(9):669-674.

RecQ Helicases: Werner Syndrome Werner Syndrome WRN gene

exonuclease helicase RQC HRDC NLS

aa 1 1432 Autosomal Recessive WRN gene Premature aging

Short Stature insertion deletion/i duplication substitution Nonsynonymous SNP Scleroderma-like Skin Changes deletion nsertion Cataracts Osteosarcoma At older age Unusual sites: Foot, ankle, Patella Other Cancers: Soft Tissue Adult Progeria sarcoma,thyroid,myeloid disorders, melanoma, meningioma

www.asianscientist.com Adapted from WRN mutation database –University of Washington- The Monnat Lab

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RecQ Helicases : Bloom Syndrome Other Syndromes

• Autosomal Recessive RAPADILINO Syndrome Diamond-Blackfan Anemia • Sensitive to sun • RecQ4 mutations • Ribosomal S19 mutations • RA: Radial Dysplasia • Rashes • Pure red cell aplasia • PA: Patella aplasia or • Sparse subcutaneous fat hypoplasia • Congenital abnormalities • DI: Diarrhea and dislocated (craniofacial, thumb,heart, • Carcinomas, lymphomas and joints Leukemias renal) • LI: Little size and limb • Osteosarcoma rate higher than malformations • Predisposition to cancer general population • NO: Long slender nose and • 6 Osteosarcoma cases Normal Intelligence reported Adpated from Genomic disorders:Gavina nd Ming-Hui

Osteosarcoma

Predisposition Sporadic

• TP53 -LFS • TP53 -30-95%  1566 patients 15.7% • RB1 -Retinoblastoma • RB1 -30%  Analysis of Germline • RecQL4- RTS Type II • RecQL4- not reported variants 12.9% • RecQ WRN- not reported  187 genes associated • RecQ Helicase WRN- Werner with single gene • RecQ BLM -not reported • RecQ –BLM-Bloom Syndrome disorders 6.4% • Pathology- same • Pathology- Conventional OS  15.7% had PPGV • Sites- metaphyses of long  80% of these were • Sites- multicentricity, unusual sites bones, pelvis cancer predisposition • Radiation association in RB1-200- • Radiation association –less Schrader et algenes 400 fold increase common JAMA Oncology 99.9% 2015 of the patients had one VUS

Summary

Osteosarcoma can be the presenting diagnosis of LFS, RTS, WS and BLM Potential value for broad germline sequencing approach in tumor-normal analysis Early detection- Better surveillance for subsequent cancers and at-risk family members

JAMA Oncol. 2016;2(1):104-111. doi:10.1001/jamaoncol.2015.5208

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Acknowledgement: Important Information Diana Mandelkar MD Ph.D Liying Zhang MD Ph.D Regarding CME/SAMs

The Online CME/Evaluations/SAMs claim process will only be available on the USCAP website until September 30, 2017.

No claims can be processed after that date!

After September 30, 2017 you will NOT be able to obtain any CME or SAMs credits for attending this meeting.

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