Whole genome sequencing and quantitative proteomics ......Topo1 938 — 6,385 1,001 KRAS_4B 424 —...

Whole genome sequencing and quantitative proteomics reveal HPV integration and HER2 overexpression in a patient with cervical cancer: Comprehensive omics analysis driving clinical treatment decisions

S. C. Benz Ph.D, J. Z. Sanborn Ph.D., N. S. Hensley B.Sc., T. Hembrough, Ph.D., C. J. Vaske Ph.D., S. Rabizadeh Ph.D., I. Royston M.D., P. Soon-Shiong M.D.

Introduction: Selection of drugs to treat patients with cancer is typically based on the anatomical site in which the tumor is located. Here we report a treatment decision for a patient with relapsed, advanced cervical cancer that was based on a comprehensive omics analysis using whole genome sequencing (WGS) combined with quantitative proteomics.

Methods: The patient was a 44-year-old female whose disease had progressed following surgery and more than 4 lines of chemotherapy. WGS was performed on the patient’s formalin-fixed, paraffin-embedded (FFPE) metastatic tumor sample and a matched-normal reference sample. Quantitative proteomics was performed on the FFPE tumor sample by Selected Reaction Monitoring Mass Spectrometry and was quantitated at the atomolar level.

Results. WGS found somatic mutations and rearrangements and reads mapping to human papillomavirus type 18 (HPV 18). Mutations more commonly found in breast cancer (ERBB2, CDH1, and CLTCL1) were noted. The HPV 18 genome was integrated into chromosome 17 in close proximity to a 7-fold amplification of the ERBB2 gene. Proteomic analysis of the FFPE tumor validated and quantitated overexpression of HER2 protein resulting from ERBB2 gene

Abstract

Acknowledgments

Sequencing Analysis Pipeline

Case Details

We would like to thank Kathryn Boorer, Ph.D. of NantHealth, for writing support.

Academic Collaborations, Research Consortiums (TCGA)

Align Sequence,

Post-Process Alignment

Simultaneous Analysis of

Tumor DNA+RNA and Normal DNA

Tumor DNA, RNA Normal

Aligned Tumor & Normal, RNA

Raw Sequences

Five3g e n o m i c sRelative Copy Number Five3g e n o m i c sAllele Fraction, LOH Five3g e n o m i c sAnnotated Germline & Somatic Small Variants

Five3g e n o m i c sStructural Variants with Split Read Refinement

Five3g e n o m i c sRNA Expression of Mutant Alleles

Sequencing Centers

amplification, with 11,322 amol/μg of tissue protein. Clinically observed ranges for breast or gastric cancer are 150-500 amol/μg, with levels above 750 amol/μg correlating with FISH-positive amplification and clinical efficacy of trastuzumab (unpublished observation). Based on these comprehensive omic findings, trastuzumab, a therapy approved for breast and gastric cancer, was administered. The patient experienced a reduction in the size of her tumor (by CT/PET) and stabilization of her disease for 5 months.

Conclusion. WGS and proteomic profiling of this patient’s disease identified, confirmed, and quantitated an appropriate target for pharmaceutical intervention. The patient presented with cervical cancer; however, the WGS analysis pointed towards a potentially causative integration of the HPV 18 genome resulting in ERBB2 amplification along with genomic mutations more commonly found in breast cancer. Proteomic analysis further validated and quantitated the HER2 expression resulting from ERBB2 gene amplification, leading to the patient’s treatment with trastuzumab. Our findings argue for the use of comprehensive omics analysis to guide decision support for personalized management of cancer care with therapies determined based on a quantitative proteomic signature, independent of anatomical tumor type.

© 2015 NantOmics. All Rights Reserved.

Methods

• 44-year-old woman with poorly differentiated cervical adenocarcinoma

• Surgery (Sep 2011): radical hysterectomy, including bilateral salpingectomy and lymphadenectomy, with

preservation of the ovaries

• Wall invasion to outer third of cervix; horizontal spread, 2.7 cm

• No lymphatic, vascular, or parametrial invasion; lymph nodes, negative

• CT/PET, biopsy (Jul 2012): multiple pelvic masses detected (SUVmax, 43.9)

• Received carboplatin/gemcitabine followed by vinorelbine/tamoxifen/gefitinib (4 doses QW) followed by carboplatin/gemcitabine (1

dose)

• Obstructive renal failure (Sep 2012): treated with ureteral stents

• Biopsy of metastatic tumor (Nov 2012): omics analysis

• Detection of HER2 amplification/overexpression

• Received trastuzumab/lapatinib

• Disease stabilization for 14 months

• CT/PET (Apr 2013): disease progression with pleural effusions and severe hydronephrosis

• Placement of nephrostomy tubes

• Received adotrastuzumab emtansine/pertuzumab/lapatinib Q3W

• CT/PET, biopsy (Jun 2013): disease progression, minimal genetic changes in tumor

• Received trastuzumab/lapatinib/vinorelbine and high-dose tamoxifen (4 weeks) followed by carboplatin/trastuzumab/

lapatinib

• CT/PET (Sep 2013): disease progression

Informed Consent • The patient provided written informed consent to perform genomic and proteomic analysis

Whole Genome Sequencing • Formalin-fixed, paraffin-embedded tumor samples and a matched-normal reference sample

• WGS was performed by Illumina (San Diego, CA)

• ~2.5 billion paired sequencing reads resulted in sequencing depths of 45.85x and 30.69x for the

tumor and normal samples, respectively

• Burrows-Wheeler Alignment Tool was used to align reads to the modified human reference

genome, HG19 (University of California, Santa Cruz, CA)

• Copy-number estimates, somatic variants, and rearrangements were determined as described

previously1

Quantitative Proteomics • Proteomic analysis was done using Selected Reaction Monitoring Mass Spectrometry (SRM/MS)

as described previously2

Gene Panel Analysis • Cancer gene panel sequencing was performed by Foundation Medicine Inc. (Cambridge, MA)

Fluorescence In situ Hybridization • Performed by Caris Life Sciences (Irving, TX)

Xenograft experiments • In immunodeficient mice were performed by Anti-Cancer Inc. (San Diego, CA)

Results

• HER2 was amplified and flanked by structural variants that map directly to the HPV 18 sequence

• Amplification appears to have resulted in ~18 copies with a minority allele count of 1, implying that the amplification event involved only one HER2 allele

• Equal number of copies of HER2 and HPV 18 • Major capsid gene, L1, is missing

• Configuration of structural variants suggest that a circular episome was formed from regions on 17q2 (including the full coding sequence of HER2) and the HPV 18 viral genome

• Exon 16 (believed to encode a transforming form of the HER2 oncogene3) was deleted in 7% of reads (16 of 238)

• Depth of coverage indicates a late event, occurring after the region was amplified

• Proteomic analysis confirmed tumor overexpression of HER2

• ~11,322 amol/μg of tissue protein (clinically observed range for non-

amplified breast or gastric cancer patients:150-500 amol/μg)

• Treatment with trastuzumab resulted in stabilization of patient’s disease

Sanborn JZ, Salama SR, Grifford M, et al. Double minute chromosomes in glioblastoma multiforme are revealed by precise reconstruction of oncogenic amplicons. Can Res. 2013;73:6036-6045.

Hembrough T, Thyparambil S, Liao WL, et al. Selected Reaction monitoring (SRM) analysis of epidermal growth factor receptor (EGFR) in formalin fixed tumor tissue. Clin Proteomics. 2012;9:5.

Castiglioni F, Tagliabue E, Campiglio M, Pupa SM, Balsari A, Menard S. Role of exon-16-deleted HER2 in breast carcinomas. Endocr Relat Cancer. 2006;13:221-232.

References

Quantitative Tissue Proteomics Assay Report

Medical Director: Robert Heaton, MD CLIA Number 21D2043150 Maryland Business License #1872

Decisions regarding care and treatment should not be based on a single test such as this test. Rather, decisions on care and treatment should be based on the independent medical judgment of the treating physician taking into consideration all available information concerning the patient’s condition, including other pathological tests, in accordance with the standard of care in a given community. This test should be regarded as investigational and for research use only. SPC-004.04 v2

Specimen Report Patient Name: Kimberly Schleef Specimen ID: HCS-12-17127/C0608 Date of Birth: 11-May-1969 Specimen Type: Peritoneal mass Gender: Female Date Received: 07-Nov-2013 Medical Record #: 26001081 Date Reported: 14-Nov-2013 Ordering Physician: Dr. Ivor Royston Study Code: ECRU_PRO20131021_32 Institution Name: UCSD Medical Center Additional Recipient: Shahrooz Rabizaheh

Images: Specimen ID# C0608 (markings indicate areas of microdissection)

The quantitative proteomic assays presented in this Report were developed by OncoPlex Diagnostics, and are based on the company’s proprietary technology described in Hembrough, T., et al., Clinical Proteomics 2012, 9:5. Where reported, limits of detection and assay ranges were calculated for each analyte using 8-point standard curves prepared in a complex proteomic matrix.

With the exception of Her2 and the LungAdenoPlex™ markers (KRT5, KRT7, TTF-1, and TP63), clinically validated abnormal ranges have not been established, however, markers below the limit of detection (LOD) can be presumed to be absent in the tumor.

Protein Target amol/µg of Tissue Protein

Assay Limit of Detection (LOD)

Observed Range: Preclinical Expression Levels

Test Result HCS-12-17127 (C0608)

E-cadherin 400 400 – 7,800 1,447 EGFR 75 200 – 16,000 292 FAK1* N/A 450 – 9,500 612 hENT1 50 72 – 1,363 206 Her21 150 150 – 26,0001 11,322

HSP90A 50 16,000 – 42,000 23,918 HSP90B 75 16,000 – 67,000 23,783

Ki67 75 100 – 200 521 KRAS_4B N/A 424 – 35,323 919

0

3000

6000

9000

12000

15000

Her2 Protein Levels (amol/μg)

Tumor samples

Patient’s Her2 Level (11,322 amol/μg)

Outlined areas indicate regions of microdissections

Gene Assay Range Test Result (amol/μg)KRT7 250 — 100,000 47,333HSP90A 16,000 — 42,000 23,918HSP90B 16,000 — 67,000 23,783Vimentin 7,000 — 100,000 16,422Her2 150 — 26,000 11,322E-cadherin 400 — 7,800 1,447Topo1 938 — 6,385 1,001KRAS_4B 424 — 35,323 919Topo2A 392 — 7,193 913Paxillin 500 — 1,200 734

Top 10 protein levels measured in patient.

Diagnosis SurgeryMetastases

DetectedObstructive

Renal FailureOmics

AnalysisStable

DiseaseDisease

ProgressionDisease

ProgressionDisease Progression,

Omics Analysis DeathMar

2012Apr

2012Jul

2012Aug 2012

Sep 2012

Nov 2012

Dec 2012

Feb 2013

Apr 2013

Jun 2013

Jul 2013

Sep 2013

??

Gem/Carbo

then

VNR/Tam/Gef

then

Gem/Carbo

T-mab/Lap T-DM1/

P-mab/Lap

Q3W

T-mab/Lap/VNR

+high-dose Tam

T-mab/Lap/

Carbo

Gem, gemcitabine; Carbo, carboplatin; VNR, vinorelbine; Tam, tamoxifen; T-mab, trastuzumab; Lap, lapatinib; T-DM1, trastuzumab emtansine; P-mab, pertuzumab.

Abbreviations:

q12chr1737.85mb +10.00kb +20.00kb +30.00kb +40.00kb +50.00kb +60.00kb +70.00kb +80.00kb +90.00kb

Inter-Chromosomal Rearrangements WG DNA

NC_001357.1NC_001357.1 NC_001357.1

Intra-Chromosomal Rearrangements WG DNA

Overall Copy Number WG DNA

0.0

7.47

Allele Fraction WG DNA

0.5

1.00

0.00Small Variants WG DNA

illumina1-viral Primary Tumor WG DNA DataPlease zoom in to view data

illumina1-viral Blood Normal WG DNA DataPlease zoom in to view data

UCSC Known Genes

MIEN1PGAP3 ERBB2 GRB7 IKZF3

COSMIC by Wellcome Trust Sanger Institute

ERBB2ERBB2ERBB2ERBB2ERBB2ERBB2ERBB2ERBB2

ERBB2 ERBB2ERBB2 ERBB2ERBB2 ERBB2ERBB2 ERBB2ERBB2 ERBB2ERBB2 ERBB2ERBB2 ERBB2ERBB2 ERBB2ERBB2 ERBB2ERBB2 ERBB2ERBB2 ERBB2

ERBB2 ERBB2 GRB7ERBB2 ERBB2 ERBB2 GRB7

ERBB2 ERBB2 ERBB2 GRB7 IKZF3ERBB2 ERBB2 ERBB2 GRB7 IKZF3

ERBB2 ERBB2 ERBB2 ERBB2 GRB7 IKZF30

134

+ 145 variants (zoom in for detail)

chrNC_001357.1+1.00kb +2.00kb +3.00kb +4.00kb +5.00kb +6.00kb +7.00kb

Inter-Chromosomal Rearrangements WG DNA17 17 17



0.0

0.00


0.5

1.00


illumina1-viral Blood Normal WG DNA Data

illumina1-viral Primary Tumor WG DNA Data

0.0

570

UCSC Known Genes

COSMIC by Wellcome Trust Sanger Institutev1 v2 v3 v4





0.0

0.00


0.5

1.00




0.0

570

UCSC Known Genes






0.0

0.00


0.5

1.00




0.0

570

UCSC Known Genes


E6E7

E1E2

E4 E5L2

L1



NC_001357.1NC_001357.1 NC_001357.1



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7.47


0.5

1.00




UCSC Known Genes








134




NC_001357.1NC_001357.1 NC_001357.1



0.0

7.47


0.5

1.00




UCSC Known Genes








134


Predicted circular episome

17q2 + HPV18

(57.3kb)

h4h1 h2 h3





0.0

0.00


0.5

1.00




0.0

570

UCSC Known Genes


C.A.

B.HPV 18Human 17q2



NC_001357.1NC_001357.1 NC_001357.1



0.0

7.47


0.5

1.00




UCSC Known Genes








134


0kb





0.0

0.00


0.5

1.00




0.0

570

UCSC Known Genes




NC_001357.1NC_001357.1 NC_001357.1



0.0

7.47


0.5

1.00




UCSC Known Genes








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Coverage

Structural Variants

Genes

HPV 18 Regions

Coverage

Structural Variants

Genes

Human 17q2 Regions

Other relevant alterations identified:Gene Alteration InformationPTPRK p.R1389* Tumor SuppressorCDH1 p.N531S Tumor SuppressorCLTCL1 LossSMARCB1 Loss Tumor SuppressorEP300 Loss Tumor SuppressorNF2 Loss Tumor Suppressor

Whole genome sequencing and quantitative proteomics ......Topo1 938 — 6,385 1,001 KRAS_4B 424 —...

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