Next-Generation
Sequencing in Pathology:
Enabling Personalized Medicine

The material is intended to provide health care providers (HCPs) with basic
information on next-generation sequencing and is for general educational
purposes only. The guide is not intended to be used to substitute for the
exercise of the HCP’s professional judgment in providing professional services.

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Molecular Pathology and Genomics
The fast-paced development of genomic sequencing technologies is revolutionizing our understanding of thecomplexities of cancer. The number of variants identified as being associated with cancer risk onset andprogression is rapidly growing. Several well-characterized genes, erbB2 receptor tyrosine kinase 2 (HER2),
epidermal growth factor receptor (EGFR), BRAF, and RAS sarcoma genes (KRAS, NRAS), are now incorporated
into molecular cancer testing procedures.1 Next-generation sequencing (NGS) is enabling the investigation of
mutations associated with cancer risk and prognosis.2 Genomics is revolutionizing the discovery and investigationof novel drug targets that are leading to earlier diagnosis and more targeted cancer therapies. Today, several
companion diagnostic devices have been cleared or approved (Table 1).3
Cancer Type

Colorectal

Non-small cell lungcancerMelanoma

BreastGene

Variant(s)Targeted Therapy

Exons 2, 3, 4

Vectibix (panitumumab)

Codons 12, 13

Erbitux (cetuximab)

NRAS

Exons 2, 3, 4

Vectibix (panitumumab)

EGFR

Exon 19 deletions
Exon 21 (L858R)
substitution

Tarceva (erlotinib)
Gilotrif (afatinib)
Iressa (gefitinib)

T790M

Tagrisso (osimertinib)

V600E

Mekinist (trametinib)
Zelboraf (vemurafenib)
Tafinlar (dabrafenib)

V600K

Mekinist (trametinib)

Amplification

Herceptin (trastuzumab)

KRAS

BRAF

erbB2 (HER2)

Table 1. A few examples of gene variants that are associated with cancer that have targeted therapies.3

Next-Generation Sequencing
NGS offers several advantages over traditional, more labor-intensive approaches such as Sanger sequencing,polymerase chain reaction (PCR) testing, fluorescence in situ hybridization (FISH), and single-gene assays. Given
the narrow focus of these molecular methods, additional testing may be required. Such sequential testing isdependent on tissue availability and may require additional biopsy procedures. In contrast, by analyzing multiple genes
and multiple samples in a single experiment, NGS can reduce the time to meaningful results.
Several organizations, including the Association for Molecular Pathology (AMP), the American College of Medical
Genetics and Genomics (ACMG), the College of American Pathologists (CAP), and the US Food and Drug
Administration (FDA), recently published guidelines, guidance documents, or recommendations for sequencing.4-6
NGS is changing the paradigm in molecular pathology, moving from clinical diagnosis alone to providing evidence
supporting treatment recommendations.

NGS Workflow in Cancer
In principle, the concept behind NGS technology is similar to Sanger sequencing. The breakthrough innovation isthat instead of sequencing a single DNA fragment, NGS extends this process across millions of fragments in a
massively parallel fashion. This method is highly scalable and can be applied to a subset of key genes (as seen intargeted panels) or to thousands of genes simultaneously (whole-exome sequencing [WES] or whole-genome
sequencing [WGS]). Prior to testing, a pathologist examines the tissue specimen to confirm the presence andpercentage of tumor cells, their viability, and cellularity. DNA is extracted from the tumor specimen, nucleotide
sequence is determined, detected variants are interpreted, and the clinical significance is reported. This process or

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Educational content provided byturnaround time (TAT) takes approximately 6 days (Figure 1).7 Recently, the AMP and the CAP jointly publishedrecommendations for analytical validation of NGS-based oncology gene panel testing to improve the quality ofsequencing and thus provide better care for patients with cancer.8

Time

6-d

Steps
Pathological
Review

NGS
Sequencing

Read
Alignment &Mutation
Detection

ClinicalReportingFigure 1. Clinical NGS testing in molecular pathology. Adapted from Macmillan Publishers Ltd: Frampton GM,
et al. Nature. 2013;31(11):1023-1033.9

NGS Applications
Compared with traditional methods, NGS offers advantages in accuracy, sensitivity, and speed that can make asignificant impact on the field of molecular pathology. NGS can be used to study many types of variations, including
structural changes, fusions, single nucleotide variants (SNVs), small indels, gene expression, and DNA methylation.10-13
Various NGS applications are available for testing cancer samples: WGS, WES, and targeted panel sequencing,each offering different advantages (Table 2).14,15
NGS
Applications

WGS

WES

Description

Determines the
DNA sequenceof an individual’s
genomeDetermines the
DNA sequence ofthe protein coding(expressed) regionsof an individual’s
genomeAdvantages

Disadvantages• Provides genetic information ofcoding and noncoding regions• Provides a comprehensive
approach• Majority of known pathologicalabnormalities are in the exome• Majority of known
pathological abnormalities
are in the exome• Data may be more difficult
to interpret• Challenges with incidental
findings• Misses variants innoncoding regions andsome structural variants• Functional consequencesof variants are more easily
understood• Challenges withincidental findings• Usually less expensive than
WGS/WES

Targeted

Determines the
DNA sequence ofspecific genes orgene regions• Focused on specific genes, sodata interpretation is easier• Little concern regarding
incidental findings• Does not provide information
on regions outside thegene panel• Can optimize gene panel tocapture difficult regions
Table 2. Comparison of NGS applications used in oncology.14

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Whole-genome sequencing
WGS can detect variants across the genome, both coding and noncoding regions, enabling the discovery ofnovel cancer-associated variants, such as SNVs, copy number changes, and structural variants. Moreover, bycomparing tumor and normal DNA, WGS can provide a comprehensive view of changes to a specific tumor
sample. However, the large amount of data generated can be more difficult to interpret, and challenges still exist
around the interpretation of incidental findings.16

Whole-exome sequencing
WES focuses on the coding regions, approximately 1% of the total genome.17,18 Up to 85% of cancer variants arefound within exons and therefore, may be easier to interpret than intronic variants. WES allows for deeper coverageof the genome, supporting identification of low frequency variants within heterogeneous tumor samples.11
Therefore, WES may sometimes be a more cost-effective approach than WGS.

Targeted panels
Targeted sequencing, the most frequently used NGS method in molecular pathology, focuses on specific genes orgene regions commonly mutated in cancer (Figure 2).14 This approach offers some benefits over WGS and WES,
including lower cost (depending on panel size), fewer incidental findings resulting in easier data interpretation, andgene panel optimization for capturing regions that can be difficult to sequence.

Choose approach
Consider patient- Tumor type
- Ease of sampling- Previous workup

Assay- Target gene panel- Whole exome- Whole genome

Sampling method- FFPE biopsy- Fresh frozen- ctDNA

Utilize results
Action- Targeted therapy- Clinical trials- Avoiding unnecessary
therapy

Check databases- Mutation hotspot- Actionable variant- Prognostic value

Bioinformatics- Align to genome- Call sequence variant- Filter artifact

Continued monitoring
Liquid biopsies- Look for increase inallele fraction ofidentified mutations

Monitor resistance- Sequence forresistance mutations

Action- Adjust therapy as
dictated by results

Figure 2. Approach for using NGS in the diagnosis and monitoring of disease in patients with cancer.
Sequencing results are interpreted into clinically actionable information, therapy is delivered, and disease iscontinually monitored. Reprinted from Gagan J, Van Allen EM. Genome Med. 2015;7(1):80. Creative Commons
Attribution 4.0 International License (https://creativecommons.org/ licenses/by/4.0/).17

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NGS for monitoring disease using circulating tumor DNA (ctDNA)NGS is being used to monitor disease by analyzing the circulating tumor DNA (ctDNA), and in some cases thecirculating tumor cells (CTCs), that can be found in the blood. ctDNA is derived from the rapid turnover of cancer
cells due to apoptosis and necrosis, resulting in the constant release of tumor-derived nucleic acids into thecirculation.19 Applications of ctDNA include genomic profiling of tumors, monitoring of therapeutic response,
detection of mutations associated with treatment resistance, and tracking of minimal residual disease (MRD)
following treatment (Figure 3).20 The preferred source of ctDNA is plasma, given its lower background level ofwild-type DNA. However, ctDNA has also been found in other biological fluids, including urine, saliva, pleural
effusions, and cerebrospinal fluid.21-24 Although tissue biopsy remains the standard of care, it reflects only a single
point in time from a single tumor site. The diversity of molecular genetic information found within each tumor andmetastases makes tissue biopsy an inadequate method for comprehensive longitudinal genomic characterization.25
Continued refinement of ctDNA technology has the potential to revolutionize the way cancer is identified andtreated, leading to earlier diagnoses, better survival rates, and improved quality of life.
DIAGNOSIS:
Genotyping cfDNA in the blood
to determine the tumor profile
RESPONSE
AND FOLLOW UP:
Analysis cfDNA and CTC for real time
monitoring of response to treatment
TUMOR EVOLUTION:
Emergence of molecular alterations
associated with resistance to therapy
MINIMALRESIDUAL DISEASE:
The presence of cfDNA or CTC in thecirculation indicates that the disease isstill present

Figure 3. Clinical applications
using cell-free-DNA (cfDNA)
and CTCs detected in blood,
the liquid biopsy. Reprinted
from Bardelli A, Pantel K.
Cancer Cell. 2017;31(2):172179, copyright 2017, withpermission from Elsevier.20

NGS for identifying biomarkers for immunotherapy using
checkpoint inhibitors
Immunotherapy represents a major paradigm shift incancer therapeutics. Anti-cytotoxic T-lymphocyte
antigen-4 (anti–CTLA-4), and anti-programmed death-1
receptor (anti–PD-1) are checkpoint inhibitors that blocktumor pathways that inactivate cytotoxic T cells (Figure 4).
Restoring the ability of the innate and adaptive immune
system to eliminate tumor cells may lead to more
durable clinical responses in a variety of tumor types,
including melanoma, non-small cell lung cancer, bladdercancer, renal cancer, and Hodgkin lymphoma.26-31 In
patients with metastatic melanoma, overall response
rates range from 12% with CTLA-4 inhibitors to 40%
with anti–PD-1 agents.32 Given the cost of
immunotherapy, biomarkers that can identify patients
who will likely respond to these treatments are needed.
Recently, 2 powerful biomarkers were identified using
NGS analysis: tumor mutational burden (TMB), andmicrosatellite instability (MSI).

Figure 4. Checkpoint-inhibiting antibodies block the interactionsof CTLA-4 and PD-1 with their ligands, resulting in reactivation ofcytotoxic T cells. Reprinted by permission from Macmillan
Publishers Ltd: Drake CG, et al. Nat Rev Clin Oncol.
2014;11(1):24-37, copyright 2014.33

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NGS analysis for tumor mutational burden (TMB)
TMB measures the number of nonsynonymous (protein changing) mutations identified per megabase of DNA. A
recent study used WES data collected from approximately 6000 cases showed a wide range of TMB across many
cancers, with lung cancer and melanoma having the highest amounts, consistent with environmental exposuresthat contribute to tumorigenesis, such as smoking and ultraviolet light (Figure 5).34,35 TMB has been analyzed as apredictive biomarker for response to checkpoint inhibitor immunotherapy. Cancers with high TMB have beenshown to be associated with improved objective response rates (ORRs), durable clinical benefit, and improved
progression-free survival (PFS).36-38

Figure 5. Graph showing the prevalence of mutations across many cancer types. Each tumor sample (dots) was
compared with the median mutation frequency (red horizontal lines). Cancer types are listed in order from the lowest
TMB prevalence (vertical axis, log scale) to the highest prevalence on the right. ALL, acute lymphoblastic leukemia;
AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia. Reprinted by permission from Macmillan
Publishers Ltd: Alexandrov LB, et al. Nature. 2013;500(7463):415-421, copyright 2013.34

NGS for detection of microsatellite instability
Microsatellites are short, 2– to 5–base pair DNA sequences that are tandemly repeated from 10 to 60 times.39
These regions are prone to base-pair mismatching during DNA replication but can be corrected by DNA mismatch
repair (MMR) proteins. If MMR is impaired, genetic hypermutability and MSI can result. For example, MSI has been
observed in approximately 15% to 20% of colorectal cancers (CRCs).40 One study compared treatment with thePD-1 inhibitor pembrolizumab for CRCs with high levels of MSI (MSI-H) vs those without MSI. MSI-H tumors
showed an ORR of 40% and disease control rate (DCR) of 90%. In contrast, patients with MSI stable tumors had
an ORR of 0% and DCR of 11%.41 Several studies have shown that NGS can enable highly accurate detection of
MSI and may have advantages over traditional PCR-based methods, including the ability to test large batches ofsamples and provide a more standardized interpretation and additional important information beyond that provided
by PCR.42,43 MSI has significant implications for tumor etiology, prognosis, therapeutic choices, and familial cancer
risk (eg, Lynch syndrome).40

Summary
Over the last decade, advances in medical genomics have elevated the understanding of cancer biology and led
to novel approaches for the diagnosis, management, and treatment of patients with cancer. The number ofhealthcare practitioners adopting NGS continues to grow, as does the recognition of the power of NGS to offer
improved sensitivity and specificity, greater genetic coverage, and the ability to use small quantities of tissue. By
enabling the simultaneous detection of multiple types of genetic alterations and the ability to pool samples during
one sequencing run, TAT for results in laboratories using NGS has decreased. In contrast to traditional moleculartechniques, NGS has the potential for more specific, individualized, patient assessment. As results from multiple
clinical trials have shown, the integration of genomic information into patient treatment plans can lead to shortened
time to diagnosis, more targeted therapeutic strategies, and ultimately, improved outcomes for patients.

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Educational content provided byReferences
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