Molecular Cytopathology

by Bin Yang, Jianyu Rao (eds.)

Molecular Cytopathology This book reviews the current applications of molecular tools in cytopathology and provides a concise handbook for those who provide care in this era of personalized medicine Specifically the text provides a comprehensive and concise review of the emerging molecular tests available clinically in different subspecialities of diagnostic pathology It reviews the current data of molecular testing already applied in cytopathology discusses some of the biomarkers with potential utility in cyto

Publisher : Springer International Publishing

Author : Bin Yang, Jianyu Rao (eds.)

ISBN : 9783319307398

Year : 2016

Language: en

File Size : 5.74 MB

Category : Used Textbooks

Bin Yang
Jianyu Rao Editors

Molecular
Cytopathology
Essentials in Cytopathology
Series Editor
Momin Siddiqui

123

Essentials in Cytopathology

Series Editor
Momin T. Siddiqui

More information about this series at http://www.springer.com/series/6996

Bin Yang • Jianyu Rao
Editors

Molecular
Cytopathology

Editors
Bin Yang, MD, PhD
Director of Molecular Cytopathology
Department of Pathology
The Cleveland Clinic
Cleveland, OH, USA

Jianyu Rao, MD
Director of Cytopathology
Department of Pathology
and Laboratory Medicine
University of California
at Los Angeles
Los Angeles, CA, USA

ISSN 1574-9053
ISSN 1574-9061 (electronic)
Essentials in Cytopathology
ISBN 978-3-319-30739-8
ISBN 978-3-319-30741-1 (eBook)
DOI 10.1007/978-3-319-30741-1
Library of Congress Control Number: 2016943830
© Springer International Publishing Switzerland 2016
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The publisher, the authors and the editors are safe to assume that the advice and
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Printed on acid-free paper
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The registered company is Springer International Publishing AG Switzerland

Preface

As beautifully articulated by Dr. Richard Demay, cytology is a
clinical practice that combines science with art. Cytology allows
one to make a diagnosis based on changes of individual cells. The
rapid turnaround time, minimum cost and resources needed (often
just a microscope is needed), and noninvasive or minimally invasive method of obtaining diagnostic material are the major advantages for cytology. As such, morphology-based cytology has been
and will likely continue to be in the forefront of clinical diagnosis
and management of various human disease conditions, particularly
cancer. While there are many great examples of cytology’s instrumental role in patient care and clinical decision-making process,
probably the most important one is cytology’s contributions to cervical cancer screening. The combination of Pap smear-based diagnosis and colposcopy-based management has helped to drastically
decrease cervical cancer incidence and mortality in the screened
population.
However, there are indisputable limitations of a morphologybased cytology practice. Morphologic evaluation is not sufficiently
capable of determining if a cell harbors the particular genetic or
epigenetic changes that are the basis for targeted therapeutic drugs.
In an era of precision medicine where more therapies and management schemes are geared toward specific molecular changes in
disease processes, additional molecular analysis must be incorporated into a morphology-based cytological diagnostic work-up.
Fortunately, cytologic material has a distinct advantage over
formalin-fixed paraffin-embedded tissue for molecular analysis,
v

vi

Preface

such as single cell based next generation sequencing (NGS), quantitative multiplex protein or exosome analysis, or nanomechanical
profile analysis. The advantage of cytologic material is that the
cells are usually complete whole cells, rather than sections of cells.
This in turn enables the precise quantitative determination of the
biochemical or molecular changes occurring within a cell. With
the advances of techniques such as NGS, microfluidic devices, and
nanotechnology, this advantage will likely become more and more
significant.
Molecular cytopathology is still in its infancy. This book is not
intended to be inclusive of all the progress or publications in the
field of molecular cytology to date, but rather to provide a reference or background that may help residents, fellows, cytotechnologists, and cytopathologists who are interested in molecular testing
in cytologic specimens. In view of the rapid progress in this area,
periodic updates will be necessary to reflect the most current
developments.
Cleveland, OH, USA
Los Angeles, CA, USA

Bin Yang, MD, PhD
Jianyu Rao, MD

Contents

1

2

3

4

5

Development and Validation of Molecular
Testing on Cytologic Specimens ..................................
Shengle Zhang and Bin Yang

1

Molecular Techniques and Methods Applied
in Cytology ....................................................................
Gilda da Cunha Santos and Mauro Ajaj Saieg

17

Potential of Next-Generation Sequencing
in Cytology ....................................................................
Xinmin Li and Bin Yang

27

HPV Testing and Molecular Biomarkers
in Cervical Cytology ....................................................
Zaibo Li and Chengquan Zhao

47

Molecular Diagnostics in Thyroid
Cytopathology ..............................................................
Robert J. Monroe and Anagh Vora

67

6

Molecular Markers in Head and Neck Cytology ......
Zaibo Li, Huaitao Yang, and Bin Yang

99

7

Molecular Biomarkers in Pulmonary Cytology ........ 121
Qing Kay Li and Bin Yang

8

Molecular Analysis of Breast Cancer
in Cytology Samples..................................................... 139
Yun Gong

vii

viii

Contents

9

Molecular Pathology of Urine Cytology..................... 153
Jordan P. Reynolds, Meenakshi Bhasin,
Neda Moatamed, and Jianyu Rao

10

Molecular Biomarkers of Pancreatobiliary
and Gastrointestinal Tract Neoplasms ....................... 171
Huaitao Yang, Gloria Zhang, and Zaibo Li

11

Molecular Biomarkers in Hematopoietic
Neoplasms ..................................................................... 187
Serge Alexanian, Zicheng Mo, and Jianyu Rao

12

Molecular Markers in Soft Tissue
and Bone Tumors ......................................................... 225
Yaxia Zhang

13

Molecular Biomarkers in Body Fluid Cytology ........ 237
Rachel Conrad, Christine Chow, and Jianyu Rao

14

Molecular Biomarkers in Prognostication
of Uveal Melanoma ...................................................... 251
Charles V. Biscotti

15 Current Status of Microfluidics-Assisted
Cytology: The Application in Molecular Cytology ..... 261
Oladunni Adeyiga, Albert J. Mach, Jianyu Rao,
and Dino Di Carlo
Index ...................................................................................... 285

Contributors

Oladunni Adeyiga, M.D. Division of Infectious Diseases,
Department of Medicine, David Geffen School of Medicine at
University of California, Los Angeles, Los Angeles, CA, USA
Department of Bioengineering, Henry Samueli School of
Engineering & Applied Science at University of California, Los
Angeles, Los Angeles, CA, USA
Nanoelectronics Research Facility, University of California, Los
Angeles, Los Angeles, CA, USA
Serge Alexanian, M.D. Department of Pathology, University of
California at Los Angeles, Los Angeles, CA, USA
Meenakshi Bhasin, M.D. Department of Pathology, University
of Southern California, Los Angeles, CA, USA
Division of Cytopathology, Department of Pathology and
Laboratory Medicine, University of California at Los Angeles,
Los Angeles, CA, USA
Charles V. Biscotti, M.D. Pathology and Laboratory Medicine
Institute, Cleveland Clinic, Cleveland, OH, USA
Dino Di Carlo, Ph.D. Department of Bioengineering, Henry
Samueli School of Engineering and Applied Science at University
of California, Los Angeles, Los Angeles, CA, USA
Nanoelectronics Research Facility, University of California, Los
Angeles, Los Angeles, CA, USA
ix

x

Contributors

California NanoSystems Institute at University of California, Los
Angeles, Los Angeles, CA, USA
Jonsson Comprehensive Cancer Center at University of California,
Los Angeles, Los Angeles, CA, USA
Christine Chow, M.D. Department of Pathology and Laboratory
Medicine, David Geffen School of Medicine, University of
California at Los Angeles, Los Angeles, CA, USA
Rachel Conrad, M.D. Department of Pathology, University of
Oklahoma Health Sciences Center, Oklahoma City, OK, USA
Gilda da Cunha Santos, M.D., Ph.D. Department of Laboratory
Medicine and Pathobiology, University of Toronto, University
Health Network, Toronto, ON, Canada
Yun Gong, M.D. Department of Pathology, University of Texas
MD Anderson Cancer Center, Houston, TX, USA
Qing Kay Li, M.D., Ph.D. Department of Pathology, The Johns
Hopkins Medical Institutions, Baltimore, MD, USA
Xinmin Li, Ph.D. Department of Pathology and Laboratory
Medicine, David Geffen School of Medicine, University of California
at Los Angeles, Los Angeles, CA, USA
Zaibo Li, M.D., Ph.D. Department of Pathology, Ohio State
University Wexner Medical Center, Columbus, OH, USA
Albert J. Mach, Ph.D. Becton, Dickinson and Company, Franklin
Lakes, NJ, USA
Zicheng Mo, M.D., Ph.D. Kaiser Permanente Medical Center
South Sacramento, Sacramento, CA, USA
Neda Moatamed, M.D. Division of Cytopathology, Department
of Pathology and Laboratory Medicine, University of California at
Los Angeles, Los Angeles, CA, USA
Robert J. Monroe, M.D., Ph.D. Veracyte, Inc., South San
Francisco, CA, USA

Contributors

xi

Jianyu Rao, M.D. Department of Pathology and Laboratory
Medicine, University of California at Los Angeles, Los Angeles,
CA, USA
Jordan P. Reynolds, M.D. Cytopathology and Genitourinary
Pathology, Pathology and Laboratory Medicine Institute, Cleveland
Clinic, Cleveland, OH, USA
Mauro Ajaj Saieg, M.D., Ph.D. Department of Pathology, Santa
Casa de Sao Paulo School of Medical Sciences, Sao Paulo, SP,
Brazil
Anagh Vora, M.D. Cellmax Life, Mountain View, CA, USA
Huaitao Yang, M.D./Ph.D, F.C.A.P. Department of Pathology
and Laboratory Medicine, University of Cincinnati Medical
Center, Cincinnati, OH, USA
Bin Yang, M.D., Ph.D. Department of Pathology, The Cleveland
Clinic, Cleveland, OH, USA
Gloria Zhang, M.D., M.P.H. Department of Pathology, Cleveland
Clinic Lerner College of Medicine, Cleveland, OH, USA
Shengle Zhang, M.D., Ph.D. Department of Pathology, SUNY
Upstate Medical University, Syracuse, NY, USA
Yaxia Zhang, M.D., Ph.D. Department of Pathology, Cleveland
Clinic Lerner College of Medicine, Cleveland, OH, USA
Chengquan Zhao, M.D. Department of Pathology, Magee
Womens Hospital, Pittsburgh, PA, USA

1

Development and Validation
of Molecular Testing on Cytologic
Specimens
Shengle Zhang and Bin Yang

Clinical Utility
Molecular genetic analyses have been increasingly performed on
cytologic specimens to facilitate management of cancer patients.
Before developing and validating a molecular assay for clinical
utility, it is important to evaluate if the assay will significantly
change the patient management, e.g., its impacts in diagnosis, risk
assessment, prognosis and prediction of therapeutic response.
Gene fusions or rearrangements associated with chromosome
translocations in neoplasm, mostly in lymphomas and soft tissue
tumors, are useful biomarkers for purpose of diagnosis owing to
their higher frequency and specificity. For example, detections of
gene fusions of BCR-ABL1 and EWS-FLI1 have been used for
diagnosis and minimal disease monitoring of CML and diagnosis
for Ewing’s tumor, respectively. Genetic alterations in epithelial or
neuroepithelial neoplasms, mostly point mutation, insertion/
deletion and amplification, are usually not applied for purpose of

S. Zhang, M.D., Ph.D. (*)
Department of Pathology, SUNY Upstate Medical University,
750 East Adams Street, UH6804C, Syracuse, NY 13210, USA
e-mail: [email protected]
B. Yang, M.D., Ph.D.
Department of Pathology, The Cleveland Clinic,
9500 Euclid Avenue, Cleveland, OH 44195, USA
e-mail: [email protected]

© Springer International Publishing Switzerland 2016
B. Yang, J. Rao (eds.), Molecular Cytopathology, Essentials
in Cytopathology 26, DOI 10.1007/978-3-319-30741-1_1

1

2

S. Zhang and B. Yang

diagnosis due to their lower frequency (<50 %) and lack of organ/
tissue specificity, but they are successfully applied for prediction
to therapeutic response and prognosis. For example, N-myc gene
amplification and 1p/19q deletions have been used for prognosis/
risk assessment of neuroblastoma and oligodendroglioma respectively, and EGFR, KRAS, and BRAF mutations for prediction of
response to biomarker-driven (targeted) therapies for lung adenocarcinoma, colon adenocarcinoma and melanoma respectively.
Selection and decision of a molecular assay may be affected by
many factors, such as official clinical guidelines for patient
management including CAP and NCCA guidelines, availability of
FDA-approved companion molecular assays for targeted therapy,
requests by clinicians for a specific gene or disease, and reimbursable molecular-based assays by insurance company.

Technical Feasibility
Cytologic specimen may contains less amount of target cells
compared with formalin-fixed paraffin-embeded (FFPE) surgical specimen. However, cytologic specimens, especially those
obtained through fine needle aspiration, are often more suitable
for molecular assays due to the high quality nucleic acids by nonformalin fixation and less fragmented genome.

Selection of Molecular Methods
In addition to considering clinical utility as initial step, several factors should be considered before conducting validation testing of a
molecular assay. They include: (1) types of genetic alteration, such
as amplification, mutation, indels, and gene fusion; (2) clinical
sensitivity and specificity; (3) accuracy, precision and detection of
low limit; (4) simplicity, associated with shorter turn-around time
and lower cost; (5) availability of tissue type, such as fresh tissue,
FFPE or cytologic specimen; (6) clinical volume and cost effective
issue.

1

Development and Validation of Molecular Testing…

3

Common genetic alterations in neoplasm include point
mutation, indels, gene fusion, amplification, aneuploidy/polysomy
and abnormal methylation. Commonly used molecular assays in
clinical lab are polymer chain reaction (PCR), reverse transcriptional PCR (RT-PCR), Florescence in situ hybridization (FISH)
and conventional (Sanger) DNA sequencing. Recently new
highthrough put molecular technologies, such as DNA/RNA
microarray, Sequenom’s MassARRAY system and next generation
sequencing (NGS) have been introduced and increasingly used in
clinical laboratories. In addition, conventional cytogenetic lab is
employing more and more new molecular technology, such as
FISH and microarray comparative genomic hybridization Testing
(array-CGH).
PCR-based assays are suitable for detection of point mutation,
small indels, gene fusions (RT-PCR), amplification, and methylation. PCR product (amplicon) is also the first step in harvesting
targeted DNA fragment for performing DNA sequencing. FISH
assays can be used for detection of gene amplification, indels, gene
break-apart (surrogate test for gene fusion), and aneuploidy.
Sequenom’s MassARRAY and next generation sequencing (NGS)
are powerful technologies and can be used to detect almost all
types of genetic alterations. Table 1.1 summarizes the selection of
molecular methods for detection of various genetic alterations
(see Fig. 1.1).

Tissue Specimen Type and Cellularity
Another important aspect of setting up molecular test is to select
tissue type. Liquid-based cytologic materials are generally good
source for both DNA and RNA isolation. Formalin fixed paraffin
embedded tissue (FFPE), either needle biopsy or cytologic cell
blocks, are generally good for DNA but suboptimal for RNA duo
to its degradation during the tissue store and process. However,
FFPE tissues frequently are only source available for molecular
testing in practice. In this situation, proper primer design for PCR
or RT-PCR with small amplicon, usually < 200 bp, is required. In
contrast, larger amplicons can be applied with not much difficulty

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