Tumor mutation load predicts durable benefit from immune checkpoint inhibitors in several cancer types. Existing methods to estimate tumor mutation load have large input DNA and extensive infrastructure requirements and are associated with delays due to shipping biopsy samples to central laboratories. We demonstrate the ability of a targeted panel with fast turn-around time and low input requirement for estimating mutation load from tumor samples to advance research in immuno-oncology.

1. Ruchi Chaudhary℥, Wolfram Jochumℑ, Izadora Demmerℑ, Dinesh Cyanam, Vinay Mittal, Nick Khazanov, Paul Williams, Warren Tom, Alice Zheng, Geoffrey Lowman, Janice Au-Young, Seth Sadis, Fiona Hyland
℥Thermo Fisher Scientific, 180 Oyster Point Boulevard, South San Francisco CA 94080 USA; ℑInstitute of Pathology, Cantonal Hospital St. Gallen, Switzerland
Ø An in-silico analysis demonstrated OncomineTM TML
panel has adequate size and appropriate targets.
ØComparison of somatic mutations on illumina exome
and Ion Torrent TML showed high overlap.
ØThe assay stratified responders and non-responders
to anti-PD1 in the retrospective study demonstrating
capability in furthering immuno-oncology research.
ØComparison on mutation load in library replicates for
FFPE and Hapmap cell line samples showed high
reproducibility.
ØThe panel pipeline produces a detailed results report
characterizing mutations consistent with
mechanisms such as UV damage and spontaneous
deamination of 5-methyl-cytosine, as well as FFPE
deamination.
The OncomineTM Tumor Mutation Load Assay is part of the OncomineTM family
of immuno-oncology solutions that enables a multi-dimensional approach to
understanding the immune environment using the Ion GeneStudio™ S5
Systems. The solution allows clinical researchers to accurately quantify somatic
mutations from as little as 20 ng of FFPE DNA in less than 3 days.
Ø A large (1.7 Mb) targeted NGS panel sufficient to support clinical research
into the genetic correlates associated with response to immunotherapies
and may replace the need for WES.
Ø A reproducible assay that can effectively separate high and low mutation
load FFPE samples.
Estimating Mutation Load from Tumor Research Samples using a Targeted Next-Generation
Sequencing Panel on Ion Torrent
Thermo Fisher Scientific • 5781 Van Allen Way • Carlsbad, CA 92008 • thermofisher.com
Ø Low input requirement (as little as 20 ng)
Ø Targeted NGS assay (1.7 Mb) for rapid and consistent results in any research laboratory
Ø 5% minimum allele frequency to capture low frequency somatic mutations
Ø Simple workflow with Ion Reporter for tumor mutation load analysis
For Research Use only. Not for use in diagnostic procedures
© 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
1. A. Snyder et al. Genetic Basis for Clinical Response to CTLA-4 Blockade in Melanoma. The New England Journal of Medicine, 2014.
2. N. Rizvi et al. Mutational landscape determines sensitivity to PD-1 blockade in non–small cell lung cancer. Science, 2015.
3. E. M. Van Allen et al. Genomic correlates of response to CTLA-4 blockade in metastatic melanoma. Science, 2015.
Figure 2. In-Silico Comparison of OncomineTM TML and Whole
Exome Sequencing (WES): WES data of 21,056 samples
downloaded from COSMIC v80. Mutations were restricted to
OncomineTM TML targets. Mutation counts by WES strongly
correlated (r2 = 0.968) with that of OncomineTM TML assay. Figure 7. Analysis Result Report: Two page, PDF analysis result
report contains analysis settings, sample information, QC metrics,
and analyses results displaying allele ratio distribution, substitution
type and context information of somatic mutations. Example report
of a Melanoma FFPE research sample.
Figure 6. Replicates of FFPE and Cell Lines: High reproducibility of
OncomineTM TML assay observed through two replicates of three
unique FFPE tumors of three cancer types (NSCLC, CRC, and
Melanoma), two FFPE normal, and one Hapmap cell line*.
Mutation Counts by Exome
SomaticMutationscovering
OncomineTMLPanel
INTRODUCTION
METHODS
ASSAY FEATURES AND PERFORMANCE RESULTS
CONCLUSION
REFERENCES
RESULTS
Figure 5. Performance of OncomineTM TML in a Retrospective
Study of NSCLC Samples with Response from Nivolumab
(anti-PD1) Treatment*: OncomineTM TML was ran on 28
NSCLC FFPE tumor samples with response annotation
(response represented partial remission after Nivolumab
treatment). Below plot represents separation of responders
(8 samples) and non-responders (20 samples).
Figure 3. Comparing Somatic Calls of OncomineTM TML and
Illumine WES: Illumina WES and Ion Torrent TML panel were
ran on a matched FFPE tumor pair; tumor-normal analysis
was performed for both sets. Overlap analysis was done after
restricting mutations to 1.2 Mb common exonic region to
discover 86.24% of Ion Torrent’s variants shared between
the two.
Tumor mutation load predicts durable benefit from
immune checkpoint inhibitors in several cancer types1-
3. Existing methods to estimate tumor mutation load
have large input DNA and extensive infrastructure
requirements and are associated with delays due to
shipping biopsy samples to central laboratories. We
demonstrate the ability of a targeted panel with fast
turn-around time and low input requirement for
estimating mutation load from tumor samples to
advance research in immuno-oncology.
We developed a integrated solution to estimate
mutation load (Mutations/Mb) from FFPE tumor
research samples. The assay utilizes a multiplex PCR-
based target enrichment panel with 1.7 Mb of genomic
coverage. The workflow requires 20 ng of input DNA
and can leverage manual or automated library and
templating on the Ion Chef. Up to eight samples can be
sequenced on an Ion 540 Chip to achieve sufficient
depth (~500x coverage) and accuracy. The analysis
pipeline utilizes a custom variant calling and germline
variant filtering to accurately quantify somatic
mutations in cancer research samples without the need
for a matched normal sample. The workflow has a 2.5-
day turnaround time with as little as 60 minutes of
hands-on time from extracted DNA to the final report.
*DNA samples were obtained from the NIGMS Human Genetic
Cell Repository at the Coriell Institute for Medical Research.
* Samples were collected and sequenced by Institute of Pathology,
Cantonal Hospital St. Gallen, Switzerland.
Figure 4. Estimate on Data from Published Study2: Clinical trial,
WES data for 31 NSCLC subjects treated with Pembrolizumab
(anti-PD1) was downloaded with response status2. WES somatic
mutation counts were restricted to OncomineTM TML targets.
Significant difference (p = 0.0057) in mutation counts of
responders and non-responders by OncomineTM TML panel
observed.
Response Status
SomaticMutationscovering
OncomineTMLPanel
Benefit No Benefit
Sample Count
Mutation Count
Abstract Control Number: 8075