Abstract
As the value of molecular testing of cancer specimens increases, the number of tests imposed on tumor specimens also increases, often in tension with the amount of tumor material available. To develop and validate molecular assays for limited specimens, there are specific concerns that must be addressed, including DNA quality, quantity, and abundance; the number of targets/ability to multiplex; and the analytical sensitivity and specificity of the assay itself. Ultimately, weighing these considerations during assay validation in the overall context of clinical utility and laboratory workflow is critical for delivering the highest level of personalized care to patients.
References
1.
Rubinstein
WS
, Maglott
DR
, Lee
JM
, Kattman
BL
, Malheiro
AJ
, Ovetsky
M
, et al. The NIH genetic testing registry: a new, centralized database of genetic tests to enable access to comprehensive information and improve transparency
. Nucleic Acids Res
. 2013
Jan
;41
(Database issue
):D925
–35
.
[PubMed]
1362-49622.
Cancer Statistics - National Cancer Institute
. https://www.cancer.gov/about-cancer/understanding/statistics. Last updated: 04/27/2018
. Last accessed: 10/20/20183.
Lindeman
NI
, Cagle
PT
, Aisner
DL
, Arcila
ME
, Beasley
MB
, Bernicker
EH
, et al. Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment With Targeted Tyrosine Kinase Inhibitors: Guideline From the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology
. J Mol Diagn
. 2018
Mar
;20
(2
):129
–59
.
[PubMed]
1525-15784.
Lindeman
NI
, Cagle
PT
, Aisner
DL
, Arcila
ME
, Beasley
MB
, Bernicker
EH
, et al. Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment With Targeted Tyrosine Kinase Inhibitors: Guideline From the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology
. J Thorac Oncol
. 2018
Mar
;13
(3
):323
–58
.
[PubMed]
1556-08645.
Lindeman
NI
, Cagle
PT
, Aisner
DL
, Arcila
ME
, Beasley
MB
, Bernicker
EH
, et al. Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment With Targeted Tyrosine Kinase Inhibitors: Guideline From the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology
. Arch Pathol Lab Med
. 2018
Mar
;142
(3
):321
–46
.
[PubMed]
0003-99856.
Hyman
DM
, Laetsch
TW
, Kummar
S
, DuBois
SG
, Farago
AF
, Pappo
AS
, et al. The efficacy of larotrectinib (LOXO-101), a selective tropomyosin receptor kinase (TRK) inhibitor, in adult and pediatric TRK fusion cancers
. J Clin Oncol
. 2017
;35
15_suppl
:LBA2501
–2501
. 0732-183X7.
Amatu
A
, Sartore-Bianchi
A
, Siena
S
. NTRK gene fusions as novel targets of cancer therapy across multiple tumour types
. ESMO Open
. 2016
Mar
;1
(2
):e000023
.
[PubMed]
2059-70298.
Boyiadzis
MM
, Kirkwood
JM
, Marshall
JL
, Pritchard
CC
, Azad
NS
, Gulley
JL
. Significance and implications of FDA approval of pembrolizumab for biomarker-defined disease
. J Immunother Cancer
. 2018
May
;6
(1
):35
.
[PubMed]
2051-14269.
Devarakonda
S
, Rotolo
F
, Tsao
MS
, Lanc
I
, Brambilla
E
, Masood
A
, et al. Tumor Mutation Burden as a Biomarker in Resected Non-Small-Cell Lung Cancer
. J Clin Oncol
. 2018
Aug
;36
(30
):JCO2018781963
.
[PubMed]
0732-183X10.
Sussman
RT
, Shaffer
S
, Azzato
EM
, DeSloover
D
, Farooqi
MS
, Meyer
A
, et al. Validation of a next-generation sequencing oncology panel optimized for low input DNA
. Cancer Genet
. 2018
Dec
;228-229
:55
–63
.
[PubMed]
2210-776211.
Rosenbaum
JN
, Bloom
R
, Forys
JT
, Hiken
J
, Armstrong
JR
, Branson
J
, et al. Genomic heterogeneity of ALK fusion breakpoints in non-small-cell lung cancer
. Mod Pathol
. 2018
May
;31
(5
):791
–808
.
[PubMed]
0893-395212.
Nikiforova
MN
, Mercurio
S
, Wald
AI
, Barbi de Moura
M
, Callenberg
K
, Santana-Santos
L
, et al. Analytical performance of the ThyroSeq v3 genomic classifier for cancer diagnosis in thyroid nodules
. Cancer
. 2018
Apr
;124
(8
):1682
–90
.
[PubMed]
0008-543X13.
Pearlstein
S
, Lahouti
AH
, Opher
E
, Nikiforov
YE
, Kuriloff
DB
. Thyroseq V3 Molecular Profiling for Tailoring the Surgical Management of Hürthle Cell Neoplasms
. Case Rep Endocrinol
. 2018
Jul
;2018
:9329035
.
[PubMed]
2090-650114.
Rivas
AM
, Nassar
A
, Zhang
J
, Casler
JD
, Chindris
AM
, Smallridge
R
, et al. ThyroSeq®V2.0 Molecular Testing: A Cost-Effective Approach for the Evaluation of Indeterminate Thyroid Nodules
. Endocr Pract
. 2018
Sep
;24
(9
):780
–8
.
[PubMed]
1530-891X15.
Young
G
, Wang
K
, He
J
, Otto
G
, Hawryluk
M
, Zwirco
Z
, et al. Clinical next-generation sequencing successfully applied to fine-needle aspirations of pulmonary and pancreatic neoplasms
. Cancer Cytopathol
. 2013
Dec
;121
(12
):688
–94
.
[PubMed]
1934-662X16.
Karnes
HE
, Duncavage
EJ
, Bernadt
CT
. Targeted next-generation sequencing using fine-needle aspirates from adenocarcinomas of the lung
. Cancer Cytopathol
. 2014
Feb
;122
(2
):104
–13
.
[PubMed]
1934-662X17.
Kanagal-Shamanna
R
, Portier
BP
, Singh
RR
, Routbort
MJ
, Aldape
KD
, Handal
BA
, et al. Next-generation sequencing-based multi-gene mutation profiling of solid tumors using fine needle aspiration samples: promises and challenges for routine clinical diagnostics
. Mod Pathol
. 2014
Feb
;27
(2
):314
–27
.
[PubMed]
0893-395218.
de Leng
WW
, Gadellaa-van Hooijdonk
CG
, Barendregt-Smouter
FA
, Koudijs
MJ
, Nijman
I
, Hinrichs
JW
, et al. Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material
. PLoS One
. 2016
Feb
;11
(2
):e0149405
.
[PubMed]
1932-620319.
Mehrotra
M
, Duose
DY
, Singh
RR
, Barkoh
BA
, Manekia
J
, Harmon
MA
, et al. Versatile ion S5XL sequencer for targeted next generation sequencing of solid tumors in a clinical laboratory
. PLoS One
. 2017
Aug
;12
(8
):e0181968
.
[PubMed]
1932-620320.
Roy-Chowdhuri
S
, Mehrotra
M
, Bolivar
AM
, Kanagal-Shamanna
R
, Barkoh
BA
, Hannigan
B
, et al. Salvaging the supernatant: next generation cytopathology for solid tumor mutation profiling
. Mod Pathol
. 2018
Jul
;31
(7
):1036
–45
.
[PubMed]
0893-395221.
Vanni
I
, Coco
S
, Truini
A
, Rusmini
M
, Dal Bello
MG
, Alama
A
, et al. Next-Generation Sequencing Workflow for NSCLC Critical Samples Using a Targeted Sequencing Approach by Ion Torrent PGM™ Platform
. Int J Mol Sci
. 2015
Dec
;16
(12
):28765
–82
.
[PubMed]
1661-659622.
Kadri
S
, Long
BC
, Mujacic
I
, Zhen
CJ
, Wurst
MN
, Sharma
S
, et al. Clinical Validation of a Next-Generation Sequencing Genomic Oncology Panel via Cross-Platform Benchmarking against Established Amplicon Sequencing Assays
. J Mol Diagn
. 2017
Jan
;19
(1
):43
–56
.
[PubMed]
1525-157823.
Jennings
LJ
, Arcila
ME
, Corless
C
, Kamel-Reid
S
, Lubin
IM
, Pfeifer
J
, et al. Guidelines for Validation of Next-Generation Sequencing-Based Oncology Panels: A Joint Consensus Recommendation of the Association for Molecular Pathology and College of American Pathologists
. J Mol Diagn
. 2017
May
;19
(3
):341
–65
.
[PubMed]
1525-157824.
Peluso
AL
, Cascone
AM
, Lucchese
L
, Cozzolino
I
, Ieni
A
, Mignogna
C
, et al. Use of FTA cards for the storage of breast carcinoma nucleic acid on fine-needle aspiration samples
. Cancer Cytopathol
. 2015
Oct
;123
(10
):582
–92
.
[PubMed]
1934-662X25.
Chen
G
, Mosier
S
, Gocke
CD
, Lin
MT
, Eshleman
JR
. Cytosine deamination is a major cause of baseline noise in next-generation sequencing
. Mol Diagn Ther
. 2014
Oct
;18
(5
):587
–93
.
[PubMed]
1177-106226.
Noguchi
M
, Furuya
S
, Takeuchi
T
, Hirohashi
S
. Modified formalin and methanol fixation methods for molecular biological and morphological analyses
. Pathol Int
. 1997
Oct
;47
(10
):685
–91
.
[PubMed]
1320-546327.
Srinivasan
M
, Sedmak
D
, Jewell
S
. Effect of fixatives and tissue processing on the content and integrity of nucleic acids
. Am J Pathol
. 2002
Dec
;161
(6
):1961
–71
.
[PubMed]
0002-944028.
Sah
S
, Chen
L
, Houghton
J
, Kemppainen
J
, Marko
AC
, Zeigler
R
, et al. Functional DNA quantification guides accurate next-generation sequencing mutation detection in formalin-fixed, paraffin-embedded tumor biopsies
. Genome Med
. 2013
Aug
;5
(8
):77
.
[PubMed]
1756-994X29.
Betz
BL
, Dixon
CA
, Weigelin
HC
, Knoepp
SM
, Roh
MH
. The use of stained cytologic direct smears for ALK gene rearrangement analysis of lung adenocarcinoma
. Cancer Cytopathol
. 2013
Sep
;121
(9
):489
–99
.
[PubMed]
1934-662X30.
Gailey
MP
, Stence
AA
, Jensen
CS
, Ma
D
. Multiplatform comparison of molecular oncology tests performed on cytology specimens and formalin-fixed, paraffin-embedded tissue
. Cancer Cytopathol
. 2015
Jan
;123
(1
):30
–9
.
[PubMed]
1934-662X31.
Treece
AL
, Montgomery
ND
, Patel
NM
, Civalier
CJ
, Dodd
LG
, Gulley
ML
, et al. FNA smears as a potential source of DNA for targeted next-generation sequencing of lung adenocarcinomas
. Cancer Cytopathol
. 2016
Jun
;124
(6
):406
–14
.
[PubMed]
1934-662X32.
Singh
VM
, Salunga
RC
, Huang
VJ
, Tran
Y
, Erlander
M
, Plumlee
P
, et al. Analysis of the effect of various decalcification agents on the quantity and quality of nucleic acid (DNA and RNA) recovered from bone biopsies
. Ann Diagn Pathol
. 2013
Aug
;17
(4
):322
–6
.
[PubMed]
1092-913433.
Higuchi
Y
. Chromosomal DNA fragmentation in apoptosis and necrosis induced by oxidative stress
. Biochem Pharmacol
. 2003
Oct
;66
(8
):1527
–35
.
[PubMed]
0006-295234.
Valpione
S
, Gremel
G
, Mundra
P
, Middlehurst
P
, Galvani
E
, Girotti
MR
, et al. Plasma total cell-free DNA (cfDNA) is a surrogate biomarker for tumour burden and a prognostic biomarker for survival in metastatic melanoma patients
. Eur J Cancer
. 2018
Jan
;88
:1
–9
.
[PubMed]
0959-804935.
Lu
T
, Li
J
. Clinical applications of urinary cell-free DNA in cancer: current insights and promising future
. Am J Cancer Res
. 2017
Nov
;7
(11
):2318
–32
.
[PubMed]
2156-697636.
Connolly
ID
, Li
Y
, Pan
W
, Johnson
E
, You
L
, Vogel
H
, et al. A pilot study on the use of cerebrospinal fluid cell-free DNA in intramedullary spinal ependymoma
. J Neurooncol
. 2017
Oct
;135
(1
):29
–36
.
[PubMed]
0167-594X37.
Gangadhar
TC
, Savitch
SL
, Yee
SS
, Xu
W
, Huang
AC
, Harmon
S
, et al. Feasibility of monitoring advanced melanoma patients using cell-free DNA from plasma
. Pigment Cell Melanoma Res
. 2018
Jan
;31
(1
):73
–81
.
[PubMed]
1755-147138.
Kis
O
, Kaedbey
R
, Chow
S
, Danesh
A
, Dowar
M
, Li
T
, et al. Circulating tumour DNA sequence analysis as an alternative to multiple myeloma bone marrow aspirates
. Nat Commun
. 2017
May
;8
:15086
.
[PubMed]
2041-172339.
Pugh
TJ
. Circulating Tumour DNA for Detecting Minimal Residual Disease in Multiple Myeloma
. Semin Hematol
. 2018
Jan
;55
(1
):38
–40
.
[PubMed]
0037-196340.
van Ginkel
JH
, Huibers
MM
, van Es
RJ
, de Bree
R
, Willems
SM
. Droplet digital PCR for detection and quantification of circulating tumor DNA in plasma of head and neck cancer patients
. BMC Cancer
. 2017
Jun
;17
(1
):428
.
[PubMed]
1471-240741.
Christensen
E
, Nordentoft
I
, Vang
S
, Birkenkamp-Demtröder
K
, Jensen
JB
, Agerbæk
M
, et al. Optimized targeted sequencing of cell-free plasma DNA from bladder cancer patients
. Sci Rep
. 2018
Jan
;8
(1
):1917
.
[PubMed]
2045-232242.
Bando
H
, Yoshino
T
, Tsuchihara
K
, Ogasawara
N
, Fuse
N
, Kojima
T
, et al. KRAS mutations detected by the amplification refractory mutation system-Scorpion assays strongly correlate with therapeutic effect of cetuximab
. Br J Cancer
. 2011
Jul
;105
(3
):403
–6
.
[PubMed]
0007-092043.
Richardson
AL
, Iglehart
JD
. BEAMing up personalized medicine: mutation detection in blood
. Clin Cancer Res
. 2012
Jun
;18
(12
):3209
–11
.
[PubMed]
1078-043244.
Jennings
L
, Van Deerlin
VM
, Gulley
ML
; College of American Pathologists Molecular Pathology Resource Committee
. Recommended principles and practices for validating clinical molecular pathology tests
. Arch Pathol Lab Med
. 2009
May
;133
(5
):743
–55
.
[PubMed]
1543-216545.
Burd
EM
. Validation of laboratory-developed molecular assays for infectious diseases
. Clin Microbiol Rev
. 2010
Jul
;23
(3
):550
–76
.
[PubMed]
0893-851246.
Durbin
RM
, Altshuler
DL
, Durbin
RM
, Abecasis
GR
, Bentley
DR
, Chakravarti
A
, et al.; Genomes Project C
. Abecasis GR, Altshuler D, Auton A, Brooks LD, Durbin RM, Gibbs RA, Hurles ME, McVean GA: A map of human genome variation from population-scale sequencing
. Nature
. 2010
;467
(7319
):1061
–73
. 0028-083647.
Zhang
W
, Ng
HW
, Shu
M
, Luo
H
, Su
Z
, Ge
W
, et al. Comparing genetic variants detected in the 1000 genomes project with SNPs determined by the International HapMap Consortium
. J Genet
. 2015
Dec
;94
(4
):731
–40
.
[PubMed]
0022-133348.
Cornish
A
, Guda
C
. A Comparison of Variant Calling Pipelines Using Genome in a Bottle as a Reference
. BioMed Res Int
. 2015
;2015
:456479
.
[PubMed]
2314-613349.
Jain
D
, Allen
TC
, Aisner
DL
, Beasley
MB
, Cagle
PT
, Capelozzi
VL
, et al. Rapid On-Site Evaluation of Endobronchial Ultrasound-Guided Transbronchial Needle Aspirations for the Diagnosis of Lung Cancer: A Perspective From Members of the Pulmonary Pathology Society
. Arch Pathol Lab Med
. 2018
Feb
;142
(2
):253
–62
.
[PubMed]
0003-998550.
Lin
O
. Telecytology for Rapid On-Site Evaluation: current Status
. J Am Soc Cytopathol
. 2018
Jan-Feb
;7
(1
):1
–6
.
[PubMed]
2213-294551.
Troncone
G
. All-in-one: the dream and reality of molecular cytopathology testing on routine lung cancer smears
. Cancer Cytopathol
. 2018
Mar
;126
(3
):155
–7
.
[PubMed]
1934-662X52.
Guseva
NV
, Jaber
O
, Stence
AA
, Sompallae
K
, Bashir
A
, Sompallae
R
, et al. Simultaneous detection of single-nucleotide variant, deletion/insertion, and fusion in lung and thyroid carcinoma using cytology specimen and an RNA-based next-generation sequencing assay
. Cancer Cytopathol
. 2018
Mar
;126
(3
):158
–69
.
[PubMed]
1934-662X53.
Peluso
AL
, Ieni
A
, Mignogna
C
, Zeppa
P
. Lymph Node Fine-Needle Cytology: Beyond Flow Cytometry
. Acta Cytol
. 2016
;60
(4
):372
–84
.
[PubMed]
0001-554754.
Aisner
DL
, Rumery
MD
, Merrick
DT
, Kondo
KL
, Nijmeh
H
, Linderman
DJ
, et al. Do More With Less: Tips and Techniques for Maximizing Small Biopsy and Cytology Specimens for Molecular and Ancillary Testing: The University of Colorado Experience
. Arch Pathol Lab Med
. 2016
Sep
;140
(11
):1206
–20
.
[PubMed]
0003-998555.
Priore
S
, Lieberman
D
, Milano
JR
, Sussman
RT
, Rosenbaum
JN
, Roth
JJ
. Abstract OTH002: Standardized Protocol for Salvaging Quality or Quantity Not Sufficient (QNS) Samples in an Academic NGS Laboratory
. J Mol Diagn
. 2018
;20
:895
–1039
.1525-1578© 2019 S. Karger AG, Basel
2019
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.
