Marilyn M. Li, M.D. Dept. Pathology and Lab Medicine Children’s Hospital of Philadelphia

CGC Annual Meeting 08-07-2017, Denver, CO

Clinical Implementation of the Standards and Guidelines for the Interpretation and Reporting of sequence variants in cancer:

The CHOP Experience

Cancer NGS Result Interpretation and Reporting

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Evidence-based Categorization

Li, et al. JMD, Vol. 19, No. 1, January 2017

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• Four large NGS panels: Solid, Heme, Fusion, & Hereditary

• All coding regions and 25 (10) bp flanking sequences

• All known intronic pathogenic variants

• CNV and LOH/AOH analyses

• Nearly 600 known fusions and many more novel fusions

Agilent QXT Lib Prep KIT

MiSeq HiSeq

LIM system Reporting

QC check

QC check

The CHOP NGS Panels

ArcherDx

HerediPanel Heme Panel Solid Panel

Fusion Panel

Home Brew Var. annotation

NextGENe Homebrew SNV/

indel/CNV

QC and confirmation

Core Technologies

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Multiple unique start sites

Knowledge of one partner of the fusion is sufficient to design primers for AMP

Anchored Multiplex PCR (AMPTM)

ROI SNPs for CNV SNPs for ID

• The CHOP Hematological Cancer Panel contains 99 (117)

genes associated with pediatric and adult hematological malignancies

• The CHOP Solid Tumor Panel contains 237 (238) genes associated with pediatric and adult solid tumors

• The panels also include all known intronic mutations associated with these genes

• The panels allow the detection of mutations (SNV and indels) and copy number variations (CNVs)

• The panels can detect certain loss of heterozygosity (LOH)

• The panels work on FFPE samples

Somatic Cancer Panels

Average depth: 1,600x

Average depth: 1,800x

• The Fusion Panel contains 106 (109) fusion partner genes and can potentially detect over 586 (>600) different fusions including novel fusions in both solid and liquid tumors

• The panel uses Archer Anchored Multiplex PCR (AMP™) technology

• The panel works on BM, fresh tissues, and FFPE tissues

Fusion Panel

Clinically Significant Variants

• A 12 year old boy with high risk Pre B-cell acute lymphoblastic leukemia (ALL)

• AALL1131/VHR standard arm A Phase III

Randomized Trial for Newly Diagnosed High Risk B-Lymphoblastic Leukemia (B-ALL)

• Day 8 MRD: 43.3% • Day 29 MRD: 40%

Example 1: Targeted Therapy Tier 2 Therapeutic Variant

46,XY,der(6)t(6;14)(p23;q32)del(6)(q15q23),der(9)inv(9)(p?q?34)del(9)(p21p13),del(9)(q22q31),der(14)t(6;14)(p23;q32)[14].ish der(6)(IGH+),der(14)(IGH)/46,XY[4]

A High Risk Pre B-cell ALL

JAK2

A High Risk Pre B-cell ALL

Identification of Novel Fusions

GENE1-JAK2 Fusion

Gene1

GENE1-JAK2 Fusion

A Novel JAK2 Fusion

1

0.61

0.02 0.011 0.0005661 0.0038416 0.0000174 0

0.2

0.4

0.6

0.8

1

1.2

M1 M2 M3 M4 M5 M6 M7

Real Time qPCR showed nearly two log reduction after

two months of ruxolitinib treatment

Gel picture of RT-PCR

3 forward primers which located at exon 8 (F3), exon 9 (F2) and exon 10 (F1) of GENE1 and 3 reverse primers which located at exon 19 (R1), exon 20 (R2) and exon 22 (R3) of JAK2 were designed.

+ruxolitinib

Monitor Fusion Transcripts

Hyperdiploid ALL Hypodiploid ALL

• High hyperdiploidy (51–67 chromosomes) is the most common cytogenetic abnormality pattern

• ISCN 2016: Hyperdiploidy – 47-57 chromosomes

• nonrandom gain of chromosomes X, 4, 6, 10, 14, 17, 18, and 21

• Favorable prognosis

EXAMPLE 2: Risk Stratification Tier 1 Prognostic Variant

• Hypodiploidy - fewer than 45 chromosomes

• High-hypodiploid - 40-45 chromosomes

• Low-hypodiploid – 31-39 chromosomes

• Near haploid – 24-30 chromosomes

• Masked hypodiploid - doubling of the chromosomal content: 50 to 78 chromosomes

• The presence of a doubled clone does not affect the prognosis of hypodiploid ALL

Hypodiploid ALL

Modal numbers of all 218 hypodiploid ALL cases with <40 chromosomes reported in the literature. There are 2 clear peaks centered on 27 and 36 chromosomes, corresponding to near-haploid and low-hypodiploid ALL, respectively

Safavi, et al. Blood 2017 129:420-423

OS for 130 evaluable, non-Ph+ patients by modal chromosome number: 44 chromosomes, 40 to 43 chromosomes, 30 to 39 chromosomes, and 24 to 29 chromosomes.

Nachman, et al. Blood 2007 110:1112-1115

Undistinguishable by Path/Cyto

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Hyperdiploid vs. Hypodiploid ALL

Hyperdiploid ALL

Doubling of Near Haploid ALL

AA AB

BB

Example 4: Precision Diagnosis Tier 2 Diagnostic Variant

• 16 yo F who presents with R mandibular mass

• Presented first to dentist,

referred to oral surgeon

• 3 teeth removed and

biopsy taken

• Biopsy sent to multiple

pathologists including

John Hopkins

Identified as malignant neoplasm only

• Sent to CHOP for further

work up and care

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• Second biopsy obtained at CHOP • Still unclear diagnosis, send to MSK for second opinion • MSKCC favored small cell osteosarcoma

• FISH returned with EWSR1 break apart • CHOP favored Ewing's sarcoma

• Started on AEWS0031-like therapy • For local control, family offered surgery or radiation • Opted for radiation

• Completed therapy 6/12/2015 • End of therapy scans with residual soft tissue mass, now more

cystic post-radiation • Follow up imaging:

• ~1 year after completion of therapy: New/increased restricted diffusion suggests disease progression

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Ending a Diagnostic Odyssey

Fusion Panel with EWSR1 – CREB3L3

EWSR1 Chr 22: Exon 7 Breakpoint

CREB3L3 Chr 19: Exon 4 Breakpoint

Sanger Confirmation

Fusion Panel with EWSR1 – CREB3L3

• Novel fusion

• CREB3L3 paralog fusions (EWSR1-CREB3L1 and EWSR1-CREB3L2) are reported in sclerosing epitheliod fibrosarcoma, small cell osteosarcoma, low-grade fibromyxoid sarcoma

• Additional pathologic workup showed tumor to be MUC4 positive, consistent with sclerosing epithelioid fibrosarcoma

MUC4 IHC

Sclerosing Epithelioid fibrosarcoma (SEF)

• Previously thought to be a low grade soft tissue sarcoma

• Recent case series revealed

• Local recurrence can be >50%

• Metastasis 40-80% (lung > bone > pleura/chest wall)

• Mortality 25-57%

• Most often in deep soft tissues of adults, but several recent case series of SEF originating from bone

• One case in the anterior mandible

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SEF - treatment

• No standard of care

• One case report of high chemotherapy resistance • Treated with osteo-

sarcoma-style therapy, 100% viable tumor at time of resection

• Some reports of persistent

remission after resection alone • Must be a wide resection or else prone to local

recurrences • Radiation has been used neoadjuvantly and adjuvantly

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Patient Current Status

• Underwent radical resection of mandible with fibular graft reconstruction

• Margins were free of tumor

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• 7 y/o male, no PMH

• One week vague symptoms of L-sided weakness • Day of admission with L facial

droop and L foot drag

• CT with R thalamic mass

• Biopsy of mass on Day 1 admission

• Pathology consistent with thalamic (WHO Grade III) anaplastic astrocytoma

• Spinal MRI with no metastatic disease

Example 4: Cancer Predisposition Tier 1B Variant

Gene Nucleotide Amino acid VAF Read depth

MSH2 c.1906G>C p.A636P 0.905 876

TP53 c.847C>T p.R283C 0.872 836

BCOR c.2256_2257del p.R752fs* 0.754 1478

FLCN c.1285del p.H429fs* 0.706 690

ATM c.1071del p.F357fs* 0.383 149

NF1 c.4600C>T p.R1534* 0.373 1329

PPM1D c.1349delT p.L450* 0.367 1332

MSH6 c.3261dup p.F1088fs* 0.36 1962

NF1 c.3739_3742del p.F1247fs* 0.349 1105

ASXL1 c.1934del p.G645fs* 0.348 943

H3F3A** c.83A>T p.K28M 0.243 1574

Tier 2 Variants of potential clinical significance

NGS Solid Panel

*Eight other rare variants of unknown significance; **Reclassified as Tier 1A

CM16-079 SolidPanel TP53 c.847C>T, p.R283C

CM16-079 SolidPanel MSH2 c.1906G>C, p.A636P

VAF 87%, Somatic? VAF 90%, Germline?

CM16-079 SolidPanel MSH6 c.3261dup,p.F1088fs*

CM16-079 SolidPanel H3F3A c.83A>T, p.K28M

Reference

Patient

NGS Solid Panel: IGV/Sanger View

Chr2: MYCN cnLOH; DNMT3A cnLOH; ALK dup; MSH2 del, MSH6 del, XPO1 del, PDCD1 del, ERBB4 dup Chr6: IRF4 amp; CCND3 del, PRDM1 del, ROS1 del, MYB del, TNFAIP3 del, ESR1 del, ARID1B del Chr11: HRAS del, MYOD1 del, WT1 del Chr17: TP53 del, AURKB del, MAP2K4 del, FLCN del

NGS Solid Panel: CNV Analysis

• Maternal grandfather: Colon cancer • MSH2 mutation, diagnosis of Lynch Syndrome

• Paternal grandfather: Colon cancer in 60’s • Paternal great-uncle: prostate cancer (50’s) • Paternal great-uncle: colon cancer (50’s) • Paternal uncle: prostate cancer (20’s)

• Informed diagnosis of a cancer predisposition syndrome • Biallelic MMR deficiency? • Li-Fraumeni syndrome?

• Preventive surveillance

• Mutations are potentially therapeutically actionable on relapse

Genomic data: Significance

Summary

• We have implemented a tier-based somatic variant categorization and reporting system based on the AMP, ACMG, ASCO and CAP guidelines

• The system is well received by oncologists, pathologists and geneticists at CHOP

• Cancer genomics is a rapidly evolving field, the clinical significance of any variant should be re-evaluated on an ongoing basis

DGD Cancer Genomic Diagnostics Fengqi Chang, Ph.D. Fumin Lin, Ph.D. Lea Surrey Minjie Luo, Ph.D. Luanne Wainwright Donna Wilmoth Adam Gleason Tammy Grou Michelle Thiess

DGD Bioinformatics Mahdi Sarmady Kajia Cao DGD Genetic Counseling Core Gozde Akgumus Other DGD members

CHOP Cancer Center Pathology Dept. Steve Hunger, M.D. John Maris, M.D. Yael Mosse, M.D. Sarah Tasian, M. D. Angela Waanders, M.D. Bruce Pawel, M.D. Mariarita Santi-Vicini, M.D. Gerald Wertheim. M.D. And others

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