Cancer Statistics

Clinical Application of Next Generation Sequencing for Personalized Medicine in Solid Tumors

Presented by: Ryan Bender, PhD FACMG

Cancer Statistics

New driver mutations in non-small-cell lung cancerWilliam Pao, Nicolas Girard, Lancet Oncol 2011; 12: 175–80

• Mutations associated with drug sensitivityEGFR Gly719X, exon 19 deletion, Leu858Arg, Leu861Gln

• Mutations associated with primary drug resistanceEGFR exon 20 insertions

• Mutations associated with acquired drug resistanceEGFR Thr790Met, Asp761Tyr, Leu747Ser, Thr854Ala

• Traditionally, non-small-cell lung cancers have been classified according to histological features.

• Various driver mutations have been associated with these cancers over time.

• The mutations are mutually exclusive, except for those in PIK3CA.

Evolution of Knowledge in NSCLC

Lung Genetic Profiles Today (by Histology)

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Next-Generation SequencingGreater volume of clinically-actionable information • Additional markers analyzed • Pathways more fully interrogated • Clinical trials opportunities expanded

Additional service and reporting options • Expanded NGS panel for Select and Comprehensive profiles • 45 gene pan-tumor NGS panel

Enhanced specimen-friendly requirements • Smaller specimens accepted (8-10 unstained, unbaked, positively charged slides) • FFPE or Formalin samples accepted

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Illumina MiSeq

©2013 Caris Life Sciences and affiliates.

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Illumina MiSeq TruSeq Amplicon Cancer Hotspot Panel Validation

• Used 80 FFPE samples (70 FFPE tissue samples, 6 cell lines and 4 HapMap samples)

• >98% accuracy when compared to Sanger. • One discordant result stemming from misalignment of

A502_Y503dup mutation in KIT (Using Pindel and validating bioinformatics solution)

• >97% precision for inter-operator, inter-lot and inter-machine tests (Most sources of discordance involved indeterminate results)

• Linear and reliable variant detection down to 5%• All reported mutations have >99% confidence at a sensitivity of

10%©2013 Caris Life Sciences and affiliates.

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NGS Use in Pathway Analysis For Personalized Medicine


Current Biomarkers for Colorectal Cancer

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Anti-EGFR Monoclonal Antibodies

©2012 Caris Life Sciences, Ltd. and Affiliates

• Cetuximab (Erbitux)– Manufactured and marketed by ImClone and Bristol-Myers

Squibb (~$30,000/ 8 week cycle)– Approved in 2006 for treatment of squamous cell

carcinomas of the head and neck • Panitumamab (Vectibix)– Manufactured by Amgen (~$100,000/year)– Approved for treatment of colorectal cancer in 2006

• 2009 – Discovered KRAS mutations are negative indicators of response to EGFR mabs in colorectal cancer

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The EGFR Pathway and Colon Cancer


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The “Real” EGFR Pathway


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The EGFR Pathway and Colon Cancer


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Benefit of Molecular Profiling on Therapy Response

©2013 Caris Life Sciences and affiliates.Adapted from DeRooke et al. 2010

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Current Commercial and Caris Offerings

Gene ASR Commercial Kits Current NGS Offering

KRAS 7 most frequent mutations affecting codons 12 and 13

All mutations at codons 12, 13 and 61 as well as additional mutations (A146T)

BRAF V600E/K All V600 mutations as well as mutations in exon 11 and other mutations near V600 (D594, L597 and K601)

PIK3CA E542K, E545K, E545D and H1047R

All mutations in exons 9 and 20 as well as select mutations in exons 1, 5 and 7

NRAS None All codon 12, 13 and 61 mutations

PTEN None Protein expression by IHC and hotspot mutation analysis


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NGS as a Companion to Companion Diagnostics


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Roche cobas BRAF Mutation Assay• FDA approved companion

diagnostic for Zelboraf use• Specific to the p.V600E

mutation• Can pick up other

mutations – p.V600K, p.V600D, p.V600E(2), V600_K601delinsD

• Does not detect p.V600R


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BRAF Mutation Analysis• Sequencing

– V600E– Other V600 mutations– Exon 15 insertions/deletions (V600_K601delinsE/D)– L597 and K601 mutations– Exon 11 mutations

• FDA Approved Method – BRAF cobas 4800 V600 mutation test– PCR based– FDA approved companion diagnostic for Zelboraf and Debrafenib


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BRAF Mutation Analysis: PCR vs Sequencing


Sample PCR Result Sequencing Result

Patient 1 Wild Type V600E(2)

Patient 2 Mutated V600K

Patient 3 Wild Type V600K

Patient 4 Wild Type V600R

Patient 5 Mutated Wild Type

V600E

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Detection of BRAF V600E(2) by NGS

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Detection of BRAF V600E(2) by NGS

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Cobas BRAF Mutation Assay

23©2012 Caris Life Sciences, Ltd. and Affiliates

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NGS and Incidental Findings


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Case #1• 64 y/o patient presenting with growth on spinal cord• Biopsy reveal heavily pigmented nodule positive for MART-1,

HMB-45 and MITF• Malignant melanoma with unknown primary favored as Dx• Sample tested for standard melanoma markers• NGS performed with a request to report BRAF and KIT

mutation status• GNA11 Q209L mutation detected


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Case #1 H&E

27©2013 Caris Life Sciences and affiliates.

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Melanoma vs Melanocytoma

Melanocytoma Melanoma

Metastatic Not typical Typical

5-yr Survival ~80% complete resection~40% incomplete resection

Stage II – 45-80%Stage IV – 5-20%

Typical Therapy Surgical resection and/or radiation

Surgical resection and/or chemotherapy

Typical Mutated Oncogenes

GNAQ and GNA11 BRAF, NRAS and KIT

Targeted Therapies None Currently Available –MEK inhibitor trials

Vemurafenib, Imatinib, Sunitinib

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Conclusions• NGS sequencing is a high throughput method for

interrogation of the mutation status of a large number of biomarkers

• Pathway analysis is expensive and laborious by previous methods – becoming standard of care for cancer therapy

• NGS adds additional information that some companion diagnostics do not provide

• Potential benefit of incidental findings to clarify diagnosis or detect rare finding


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Cancer Statistics