the chances of uncovering clinically relevant

alterations.

- They require more tumor tissue, cost more

for data analysis and storage, and entail more

complex interpretation.

• FDA-approved liquid biopsy using circulating

cell-free DNA is an excellent tool when tumor

tissue is inaccessible or limited (Nat Rev Clin

Oncol 2021;18:297). While liquid biopsy has a

faster turnaround time, it has a higher false-

negative rate than standard biopsy NGS.

NON-SMALL CELL LUNG

CARCINOMA (NSCLC)

• Molecular proling is used to predict therapeu-

tic efcacies in NSCLCs and typically includes

EGFR, KRAS, ROS1, ALK, MET, BRAF, RET,

ERBB2 (HER2), and NTRK (Crit Rev Oncol

Hematol 2021;157:103194). Also useful is

PD-L1 (programmed cell death ligand-1) by

immunohistochemistry (IHC) to predict

response to immune checkpoint inhibitors.

• EGFR mutations involve 10%-35% of lung

adenocarcinoma in Western populations (and as

high as 50% in Asian populations) and 3% of

lung squamous cell carcinoma (SqCC). Muta-

tions can predict therapeutic response to EGFR-

specic tyrosine kinase inhibitors (TKIs).

- Acquired resistance inevitably develops,

mostly commonly due to EGFR T790M

missense mutation, accounting for ~50% of

acquired resistance mutations. FDA-approved

EGFR-inhibiting drugs that specically

target T790M or exon 20 insertion mutation

are available.

- MET amplication (by FISH or NGS) is

responsible for 5%-20% of anti-EGFR

resistance.

• KRAS mutations are detectable in 25%-35% of

lung adenocarcinoma, and 5% of SqCC.

Activating mutations in codons 12 and 13, and,

less commonly, codon 61, predict unfavorable

prognosis and EGFR-TKI resistance. A KRAS

inhibitor drug that specically targets the

G12C missense mutation (seen in ~13% of

Issue 16 || November 2021

WHAT’S NEW IN

MOLECULAR GENETIC

PATHOLOGY 2021:

SOLID TUMORS AND

NGS PANEL SELECTION

Guoli Chen and Patricia C. Tsang

Geisinger Medical Laboratories, Geisinger Health, Danville

and Wilkes-Barre, PA, USA

Corresponding Author: Patricia C. Tsang, MD

Geisinger Medical Laboratories, Geisinger Health,

Wilkes-Barre, PA, USA

E-mail: patctsang@hotmail.com

ORCID

Guoli Chen

https://orcid.org/0000-0002-4426-6156

Patricia C. Tsang

https://orcid.org/0000-0003-0523-9793

Abstract

The linchpin of precision medicine is molecular

genetic and genomic testing. Molecular biomark-

ers are important for establishing precise diagno-

ses and for predicting therapeutic responses that

enable cancer patients to receive personalized and

targeted treatment. Below are highlights of the

current considerations in next generation

sequencing (NGS) panel selection, and in

molecular testing of solid tumors of the lung,

digestive system, thyroid and soft tissue.

NEXT GENERATION SEQUENCING

PANEL SELECTION

• The use of targeted NGS panels (comprising up

to several hundred genes) is more common than

whole exome sequencing (WES) in clinical

practice. Pros and cons of WES or large

targeted panels compared to small hotspot

panels include the following:

- Comprehensive tumor proling maximizes

NSCLCs) is available.

• ALK activating rearrangements occur in ~5%

of lung adenocarcinoma, particularly in young

non-smokers. The most common fusion gene

product is EML4-ALK, which predicts response

to ALK inhibitors. FISH is the gold standard

for testing, but IHC, RT-PCR and NGS can

also be used.

• ROS1 gene fusion (1%-2% of NSCLCs)

signies response to certain ALK inhibitors due

to homology between the rearranged ROS1 and

ALK genes.

• MET exon 14 skipping mutations, BRAF

V600E mutation, RET gene fusion and NTRK

gene fusion can predict therapeutic response to

their respective inhibitors.

COLORECTAL CARCINOMA (CRC)

AND CHOLANGIOCARCINOMA

• EGFR targeted treatment is effective in the

absence of KRAS and NRAS mutations.

Mutational analyses of KRAS and NRAS genes

should include codons 12, 13, 59, 61, 117 and

146. Other drug resistance biomarkers include

BRAF mutations and PIK3CA/PTEN deregu-

lation.

• Despite its low prevalence in CRC (2-3%),

ERBB2 (HER2) amplication is emerging as a

potential therapeutic target (Lancet Oncol

2021;22:779). Furthermore, activation of

ERBB2 (HER2) signaling causes resistance to

anti-EGFR therapy in a subset of patients with

metastatic CRC.

• NTRK fusions can be evaluated in CRC due to

the availability of targeted therapies.

• FGFR gene fusion is detected in ~10% of

intrahepatic cholangiocarcinoma for which two

FGFR-targeted tyrosine kinase inhibitors have

been approved - pemigatinib and ingratinib.

THYROID CARCINOMA

• Papillary thyroid carcinoma may harbor BRAF

mutations (especially V600E), TERT promoter

WHAT’S NEW

IN PATHOLOGY?

PathologyOutlines.com

Sponsored by an unrestricted grant from Geisinger Medical Laboratories: Dedicated to Laboratory Excellence

mutations, and, less commonly, RET/PTC1/2/3

rearrangements and RAS mutations.

• PAX8-PPAR

fusion, RAS mutations, and

TERT mutations are enriched in follicular

thyroid carcinoma, while BRAF V600E is

unusual. RAS mutations are also seen in a

minority of benign follicular adenomas.

• More than 60% of medullary thyroid carcino-

mas show somatic RET mutations.

• Anaplastic thyroid carcinomas can harbor

TERT, TP53 and/or BRAF mutations.

• Preoperative molecular testing is valuable in

thyroid nodules with indeterminate FNA

cytology. The gene panel typically includes

TERT, BRAF, PAX8/PPAR

, RAS, RET/PTC

and TP53. Below are three commonly used

commercial platforms:

- Thyroseq

involves next-generation DNA

and RNA sequencing of 112 genes to stratify

thyroid nodules as likely benign or likely

malignant.

- Arma

Gene Sequencing Classier is an

RNA-based test. Similar to ThyroSeq

, it is

mostly a rule-out assay with acceptable

rule-in capability.

- ThyGeNEXT

/ThyraMIR

uses a combina-

tion of two tests. If no mutation is found in

the rst panel by DNA and RNA sequenc-

ing, another test is performed using micro-

RNA expression.

BONE AND SOFT TISSUE TUMORS

• Molecular testing has led to the discovery of

new mesenchymal tumor entities. For example,

CIC-DUX4 sarcoma is a recently described

small round blue cell tumor associated with

more aggressive disease than Ewing sarcoma

with characteristic EWSR1-FLI1 fusion.

Immunohistochemical detection of ETV4, a

transcriptional target of CIC-DUX4, is a useful

diagnostic tool.

• Amplication of the MDM2 gene detectable

by IHC/FISH/NGS is helpful for conrming a

diagnosis of atypical lipomatous tumor/

well-differentiated liposarcoma (~93%) or

dedifferentiated liposarcoma (~97%). The

oncogenicity of MDM2 is related to TP53 inac-

tivation (Fig. 1).

• Gene fusion testing by FISH or NGS is helpful

for classifying rhabdomyosarcomas (Pediatr

Dev Pathol 2021;24:395).

- Recurrent gene fusions are detected in ~80%

of alveolar rhabdomyosarcoma (ARMS), and

involve mostly the FOXO1 gene fusing with

either PAX3 or PAX7. Fusion positive

ARMS is associated with a worse prognosis

than fusion negative ARMS.

- Embryonal rhabdomyosarcoma (ERMS)

characteristically lacks gene fusions, but

shows chromosomal losses/gains and gene

mutations. ERMS is similar to fusion

negative ARMS in clinical behavior.

- Most congenital/infantile spindle cell and

sclerosing rhabdomyosarcomas show NCOA2

in addition to VGLL2 gene fusions. This

entity is associated with a favorable progno-

sis. However, patients who harbor MYOD1

mutations tend to have aggressive disease.

Dr. Chen is an Associate and Attending

pathologist at Geisinger Medical Center,

where he participates in clinical, academic and

teaching services in surgical pathology and

molecular diagnostics. He has actively

published and lectured on topics related to

molecular pathology of solid tumors.

Dr. Tsang joined the PathologyOutlines

editorial board in 2019 and has been its

Deputy Editor-in-Chief for Clinical Pathology

since 2020. She is a Clinical Associate

Professor and Laboratory Medical Director in

Geisinger’s Northeast Region and has been a

practicing molecular pathologist for more

than a decade.

Meet the Authors

Fig. 1. Dedifferentiated liposarcoma with neoplastic spindle cells in perigastric mass. (A) H&E stain, 40 x. (B)

FISH shows abnormally ampliﬁed

MDM2

gene (orange signals) relative to CEP12 control (green).

MDM2

CEP12 ratio > 2.0:1 normal cut-off.