Molecular testing techniques in cytology specimens (FNAC, Body fluids, exfoliate cytology)

1. MOLECULAR TESTING TECHNIQUES
IN CYTOLOGY SPECIMENS
– SUDIPTA NASKAR
MODERATOR – DR. VINOD K. ARORA

2. INTRODUCTION

3.  Molecular cytopathology is defined as the application of
molecular studies to any type of cytology specimen, whether
gynecological, exfoliative, or fine-needle aspiration cytology.
• Molecular genetic analyses have been increasingly performed on
cytological specimens to facilitate management of cancer
patients.
• Advances in minimally invasive interventional procedures have
resulted in an increasing reliance on small biopsies along with
exfoliative and fine-needle aspiration (FNA) cytology specimens
not only for diagnostic purposes
• but also for ancillary molecular testing to guide the increasingly
“personalized” management of patients with cancer.

4.  Recognition of tumor heterogeneity at the molecular level
are in increasing demand for disease like thyroid neoplasms,
lung CAs, Urothelial carcinoma etc.
 Rapid development of new high-throughput & multiplex
molecular testing modalities holds the promise to address
the issue of simultaneously testing for a multitude of
different genomic abnormalities using cytology samples.

5. Technical Feasibility
 Cytologic specimen contains less amount of target
cells
 However, cytological specimens, especially those
obtained through fine needle aspiration, are often
more suitable for molecular assays due to the high
quality nucleic acids by non-formalin fixation and
less fragmented genome.

6. • Considering clinical utility as initial step, following
factors should be considered before conducting
validation testing of a molecular assay.
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 all smears , cell
blocks or fresh cytological specimen (Either FNA or
Exfoliative)
6. Clinical volume and cost effective issue.

7. CYTOLOGY SPECIMEN
TYPES

8.  Fresh fine needle aspirate (Radiology guided /
unguided)
 Prepared slides from FNA
 Cell block
 Body fluids
 Effusion fluids
 Exfoliative cell materials
 Sputum
 BAL

9. Microdissection
and mutational
analysis
in
Supernatant
(normally
discarded)
Mutational
analysisCell Block
Needle
washing
Cytology slides

10. Commonly used molecular assays
 Florescence in situ hybridization (FISH)
 NucleicAcid Hybridization
 Polymer chain reaction (PCR),
 Reverse transcriptional PCR (RT-PCR)
 Next generation sequencing (NGS)
 DNA/RNA microarray
 Sequenom’s MassARRAY system
 Conventional (Sanger) DNA sequencing

11. • Fluorescence in situ hybridization (FISH) is a technique that is well
suited for detecting many types of genomic abnormalities in
cytology specimens.
• FISH involves hybridization of fluorescently labeled nucleic acid
sequences (probes) to complementary nucleic acid sequences
(targets) which are typically in the form of metaphase
chromosomes obtained from cultured, dividing cells or
decondensed chromosomes within interphase nuclei of
nondividing cells.
• FISH allows visualization of the physical location of the probe(s)
to their target(s) and can be used to interrogate specific areas of
the genome.

12. a) Enumeration Probe
– used to detect genomic gains and losses.
– Centromere probes,which are composed of alpha satellite DNA
used for detecting whole-chromosome aneuploidies
– locus−/gene-specific (unique sequence) probes are
commonly used used to detect loss or gain/amplification
of a region of interest.
b) Break-apart probes
– are optimal for assessing rearrangements of a gene that
could have more than one translocation partner, such as
MLL (KMT2A)
c) Fusion probes
are most frequently employed when fusions involve
consistent gene partners, such as BCR/ABL1

13. (a) Enumeration probe set consisting of a locus-specific probe (red) and a
centromere-specific probe (green) on the same chromosome.

14. (b) Dual fusion probe strategy consisting of two differentially
labeled, locus-specific probes that span the genes involved in a
fusion event.
(c) Break-apart probe strategy consisting of two differentially
labeled probes that flank the breakpoint (dashed lines) of one
of the gene partners involved in a fusion.

15. A wide range of cytologic preparations can be used for
FISH,
•touch preparations,
•unstained cytological smears,
•archival stained cytology slides,
•Cytospin preparations,
•liquid-based cytological preparations,
•formalin or alcohol-fixed paraffin-embedded cell
blocks

16. Preparation type Advantages Disadvantages
FFPE cell
block
1. Allows for
correlation with
adjacent H&E- or
IHC-stained section
2. Preservation of
morphology and
partial tissue
architecture
3. Cell blocks made
from aspirates may
be enriched for
tumor cells
1. Probe signal loss due
to nuclear truncation
from sectioning
2. Overlapping nuclei
can cause difficulty
scoring signals
3. May have
autofluorescent
background that
obscures signals
4. May have
inadequate tumor
cellularity

17. Preparation type Advantages Disadvantages
Cytology
smear
1. No nuclear
truncation artifact
2. Previously stained
slides can be used
3. Only technique
that allows rapid
on-site
determination of
specimen
adequacy at time
of procedure
4. Smears made from
aspirates may be
enriched in tumor
cells
1. Nuclear distortion
due to crushing
during slide
preparation
2. Overlapping nuclei
can cause difficulty
scoring signals
3. May have
autofluorescent
background that
obscures signals

18. Preparation type Advantages Disadvantages
Liquid-based
cytology
1. No nuclear
truncation artifact
2. Thin, monolayer
preparation
minimizing nuclear
overlap
3. Less background
than other
preparations
4. Less hands-on time
than many other
preparations
5. Cells are
concentrated in a
smaller area
1. More expensive than
other cytologic
preparations
2. Special
instrumentation
needed for slide
preparation
3. May have
inadequate
cellularity

19. Preparation type Advantages Disadvantages
Liquid-based
cytology
1. No nuclear
truncation artifact
2. Thin, monolayer
preparation
minimizing nuclear
overlap
3. Less background
than other
preparations
4. Less hands-on time
than many other
preparations
5. Cells are
concentrated in a
smaller area
1. More expensive than
other cytologic
preparations
2. Special
instrumentation
needed for slide
preparation
3. May have
inadequate
cellularity

20. Applications in Specific lesions

21. • Urine cytology
• Breast cytology (ERBB2)
• B-cell non-Hodgkin lymphoma (ALK and ROS1
rearrangements, t(11;14) for Mantle Cell
Lymphoma)
• Metastatic Lung Carcinoma(ALK arrangements)

22. FISH IN UROTHELIAL CELL
CARCINOMA

23. • Traditionally UCC is detected and monitored
by the combination of cystoscopy and urine
cytology tests
• Cystoscopy is an expensive and invasive
procedure and often may miss a flat lesion,
whereas urinary cytology, though noninvasive
has very low sensitivity (between 20 and 50
%) for low-grade papillary tumors.
• Adjunct molecular markers with high accuracy
for the detection of both low and high grades
of urothelial carcinoma is necessary.

24. • Two commercially available cellular-based
tests –
– more widely used UroVysion or fluorescence in
situ hybridization (FISH) test and
– ImmunoCyt or uCyt test

25. • Cytogenetically, urothelial cell carcinoma,
especially high grade UCC is an aneuploid
cancer, and contains multiple copies of
chromosomes
• Centromere enumeration probes for
chromosome 3, 7, and 17 label the
centromere of each respective chromosome.
• Presence of more than two signals within a
cell would indicate an abnormal DNA content
and increase the suspicion for malignancy.

26. DAPI stain (4′6-diamidino-2-phenylindole) is used to
stain the nucleus blue under fluorescence microscopy
Benign urothelial
cells will show a
homogeneous
staining pattern,
Malignant cells
show large nuclei
and a clumped,
heterogeneous
chromatin pattern
(reflects an aneuploid cell with dark, coarse
chromatin distribution and nuclear irregularity)

27. • Centromere enumeration probes (CEP)
directed toward the chromocenter of
chromosomes 3 (red), 7 (green), and 17
(aqua) reflect the number of copies of
chromosomes.
• Screening for large (presence of 25 urothelial cells
may be accepted in most cases) abnormal cells on
DAPI, then examining them with each filter
• When one cell with an abnormal number of
probes is detected, a search for at least 4 or
more abnormal cells should be performed

28.  Once four cells are found, the
case may be signed out as “Positive
for aneusomy”.
 These patients are at increased
risk for cancer, even when the
cytology is negative.
 An abnormal cell shows more
than 2 signals in 2 or more
probes.
 True 9p21 (gold) loss occurs in
clusters of urothelial cells which
may represent low grade papillary
lesions of the bladder.

29. FISH in Breast Cytology

30. The use of cytological smears for FISH testing as an
advantage over the use of FFPE section in that
tumor cells on smears are mostly monolayered,
It facilitates enumerating all the HER2 signals
within an entire nucleus without a truncating
artifact.
When collecting a cytology specimen that is highly
suspicious for breast cancer, collecting a sample in
formalin or saving air-dried direct smears for HER2 ISH
testing should be considered.

31. HER2 FISH can be
performed by either
a single-probe assay
or dual-probe assay.
HER2 is considered
amplified when the
assay shows ≥ 6.0
HER2 signals/cell
using a single-probe
system.
By dual-probe testing, it is positive if there is a
HER2/CEP17 ratio of ≥ 2.0 with an average HER2 copy
number of ≥ 4.0

32. Limitations of FISH

33. • As with any technology, FISH has its limitations,
and there are many examples of cases in which
FISH results are negative
• False-negative FISH results can be due to a variety
of variables, such as failure to score tumor nuclei
or atypical rearrangements.
• Complex rearrangements, or submicroscopic
insertions of part of a gene into another gene,
have been documented to result in fusions that
yield false-negative FISH results.
• Aberrant molecular results can also be
encountered even if known fusion partners with
unusual breakpoints are involved

34. Polymerase Chain Reaction Based
Assays

35. The traditional PCR assay involves 3 main steps –
1. DNA denaturation,
2. annealing, and
3. extension,
with the use of primers (sequence of nucleotides complementary
to the target DNA)
A forward and reverse primer flank the designated area
containing the desired sequence of DNA to be amplified
Multiple copies (amplicons) of the target DNA (known sequence to
verify the presence of mutations) are generated.
The amplified DNA can then be sequenced for the verification of
any mutations.

36. Cytology samples provide high-quality DNA, sufficient for a wide
array of DNA-based sequencing assays, including next-generation
sequencing (NGS)
This novel high-throughput technology represents an evolution
of conventional DNA sequencing methodologies, such as Sanger
sequencing and pyrosequencing
Sanger sequencing has long been the gold standard for the
identification of point mutations, deletions, and small insertions.
But with the advancement of NGS it is now becoming obsolete
nowadays.

37. Direct sequencing is a simple technique that uses chemically
modifi ed nucleotides labeled with distinct fluorescent dyes
a chemically modified nucleotide (dideoxynucleotide)
terminates the extension of the DNA strand at the point
of incorporation.
This results in a mixture of DNA fragments of varying
lengths. Each dideoxynucleotide, (A, T, C, or G) is labeled
with a different fluorescent dye (dye terminator).
The newly synthesized and labeled DNA fragments are
sequentially separated by size through capillary gel
electrophoresis.

38. The fluorescence is detected by an automated sequence
analyzer, and the order of nucleotides (base calling) in the
target DNA is visualized as a sequence electropherogram
In case a mutation is present, two different overlapping
peaks will be seen (from the wild-type and mutant cells).
Similarly, a deletion will be seen as a “truncation” of the
peak signals.

39. Sequencing
methods
Sanger
sequencing
NGS
Yield (MB/Run) 0.06 MB 600GB–1.8TB
Cost ($$$/MB) $1500 $0.04–$0.007
Speed (per human
genome)
13 years 2–3 days
Amount of DNA
required
500–5000 ng 10–1000 ng
Sequencing sensitivity >20 % mutation rate >1 % mutation rate
Multiplexing capability Single Multiple

40. • Data suggest that NGS can be reliably applied on cytology specimens
with high sensitivity, specificity and reproducibility
• Some of the NGS systems are –
1. Roche 454 – first commercially successful next generation
system; uses pyrosequencing technology.
2. AB SOLiD (Sequencing by Oligo Ligation Detection) System –
adopts the technology of two-base sequencing based on ligation
sequencing.
3. Illumina GA/HiSeq System –adopts the technology of
sequencing by synthesis (SBS). HiSeq 2000 uses two lasers and
four filters to detect four types of nucleotide (A,T, G, and C)
4. Compact PGM (Personal Genome Machine)
Sequencers – uses semiconductor sequencing technology.
PGM is the first commercial sequencing machine that
does not require fluorescence and camera scanning, resulting in
higher speed, lower cost, and smaller instrument size

41. • PCR based assays are the methods of choice for procurement
of mutations in
– EGFR in lung cancer
– BRAF in melanomas and papillary thyroid carcinomas
– KRAS in colon cancer.
• All sorts of cytological samples, including fresh cell
suspensions, smears (stained and unstained), cytospins and
FFPE cell blocks can be used to extract DNA for PCR based
assays.

42. Potential Applications of NGS in Cytology
• Recent improvements of FNA procedures and technological
advancement in making DNA library with the small amount of
DNA have made NGS technology applicable to cytology
specimens in clinical setting
• In Lung Carcinoma :
– The majority of NSCLC are diagnosed at an advanced stage, and missed
the best surgery time.
– Therefore, the diagnosis and therapeutic decision for lung cancer
heavily rely on minimally invasive procedures, like cytology samples

43. • Thyroid
– In approximately 25 % of thyroid nodules, the diagnosis
cannot be established and consequently classified as
indeterminate (Bethesda Cat III) by FNA cytology, hampering
clinical management of these patients
– Because some molecular markers (BRAF, NRAS, KRAS, and
PTEN) are highly specific in thyroid cancer, NGS offers the
potential to improve the accuracy of cancer diagnosis and
prognosis in thyroid nodules.
• Pancreatic Cancer
– The combination of cytological evaluation and tumor marker
mutation analysis (oncogene KRAS and tumor suppressor genes
CDKN2A/p16, SAMD4, and TP53), especially for inconclusive
cases, can potentially enhance the diagnostic power.

44.  Various alternatives to classic PCR followed by direct sequencing have been
used for cytological samples
1. Real time-PCR –
 uses oligonucleotide primers that bind specifically to flank regions
of the most common mutations.
2. High-resolution melting analysis (HRMA) –
 A rapid and cost-effective method that relies on the combination of
real time PCR and evaluation of DNA melting curves
3. Restriction fragment length analysis(RFLP)
 uses mutation-specific restriction endonucleases, and amplification is
only possible in the mutated sites.
4. MASS-Array spectrometry
 uses multiplex mutation analysis with preset commercial panels, such
as the Oncocarta Panel, have been successfully used in FNA
specimens, with reliable results.

45. DNA / RNA Microarray

47. Human Papillomavirus (HPV) Testing on
Cervical Cytology Specimens

48. Recommendations for using HPV testing for
cervical cancer prevention
 Age-based recommendation
 <20 or >65 years of age: no screening
 21–29 years of age: cytology alone every 3 years with an option of
reflex HPV testing for women withASCUS
 30–65 years of age: HPV co-screening along with Pap test every
 5 years
• In conjunction with Pap cytology
– ASCUS (21 years and older)
– Pap/HPV+ co-testing results: HPV16/18 genotyping
– LSIL in postmenopausal women
– Post-colposcopy management of women withAGC orASC-H
– Post-colposcopy management of women 21 years or older with
– ASCUS or LSIL
– Post treatment surveillance

49.  Primary HPV screening
 25–65 years of age: primary HPV screening every 5 years, if
tested negative
 HPV16/18 genotyping test, colposcopy referral if positive
 Non-HPV16/18 positive (12 hrHPV genotypes)
 Pap cytology triage

50. • To date, a total of seven commercially available HPV testing
assays under five commercial brand names were approved by
US FDA for cervical cancer screening –
1. Hybrid Capture 2 (HC2, Qiagen,Valencia, CA)
2. Cervista HPV HR and Cervista HPV16/18
3. Cobas HPV assay
4. Aptima HPV andAptima HPV16 18/45 assays
5. BD Onclarity HPV assay

51.  All the assays use liquid based preparations
 To date, the FDA approved most of HPV testing assays
specifically in ThinPrep Pap cytology
 In 2016 and 2018, FDA approved Cobas HPV assay and BD
Onclarity HPV assay for SurePath Pap specimen, respectively.
 Although bothThinPrep and SurePath Pap tests are FDA-
approved for cervical cancer screening, SurePath Pap cytology
showed a significantly lower unsatisfactory rate thanThinPrep
for Pap cytology testing.

52. Hybrid
Capture 2
Cervista
HPV
Aptima
HPV
Cobas
HPV
BD
Onclarity
HPV
PCR-based No No Yes Yes Yes
Amplification Signal Signal E6, E7
RNA
E6, E7
DNA
E6, E7
DNA
HPV
detection
13 types 14 types 14 types 14 types 14 types
HPV
genotyping
No HPV16,
18
HPV16, 18,
45
HPV16, 18 HPV16,18,
45
Internal
controls
No Yes Yes Yes Yes
Equivocal
Zone
Yes No No No No
Company Qiagen Hologic Hologic Roche Becton,
Dickinson

53. What Is the Role of Molecular
Testing?
Lung Cancer

54. • Lung cancer is the leading cause of cancer-related
death worldwide.
• diagnosis and therapeutic decision for lung cancer
heavily rely on minimally invasive procedures, either
small biopsies or cytology samples.
• Lung cancer has the most available targeted therapies.
• The targeted genes include EGFR, BRAF, KRAS, ALK, and
ROS1 . Many more potential targets, such as PIK3CA,
FGFR1, and DDR2 , are in clinical trials. Therefore, the
number of predictive biomarkers for novel targeted
drugs entering into clinical practice is expected to
rapidly increase.

55. Types of Specimens
 FNA
 In past 5 years, EBUS-TBNA has become the most popular
approach for clinical staging and obtaining cytological materials
for diagnosis and molecular testing
 Recent studies have demonstrated that cell block preparations
from FNA and other specimens, such as BAL and pleural
effusions, are superb for molecular testing.
Transthoracic FNA
EBUS-TBNA
•BAL
•Sputum
•Pleural Effusion
•Exhaled breath
condensate (EBC)

56. • Major Concerns Extracting nuclear material
– Contamination from
• blood cells,
• Inflammatory cells,
• plasma
• stromal cells
• This problem can be overcome by
– Isolating tumor cells by laser capture microdissection
(LCM)
– Microdissection of tumor tissue using microscope and
analyses potential biomarkers from selective areas of the
tumor

57.  A NIH pathology group showed that as a few as 50 tumor
cells microdissected from cell block can be used for the
detection of EGFR and K-RAS mutational testings.
 Recent studies/data from the Cleveland Clinic Foundation
have demonstrated that leftover cytolyte from the Thinprep
preparation maybe an alternative choice for both
conventional and next-generation sequencing based
mutational assays.
 In a study Ion PGM sequencing technology was applied for
targeted gene mutations analysis for 38 lung adenocarcinomas
cytology specimens
– Of the 38 specimens, 36 cases were successfully sequenced (95
%). 24/36 cases identified at least one mutations

58. Current tiered framework for molecular testing
targets in NSCLC

59. Thyroid Neoplasms

60.  Thyroid cancer typically occurs in thyroid nodules. FNA
followed by cytological examination is an accurate and cost
effective diagnostic method for evaluating thyroid nodules.
 This commonly used approach allows detecting cancer or
establishing a diagnosis of a benign nodule in most cases.
 However, in approximately 25 % of nodules, the diagnosis
cannot be established and classified as Cat III/IV/V by FNA
cytology, hampering clinical management of these patients.
 Because some molecular markers are highly specific in
thyroid cancer, NGS offers the potential to improve the
accuracy of cancer diagnosis & prognosis in thyroid nodules.

61. What Is the Role of Molecular
Testing inThyroid Cytology?

63. • Ancillary molecular testing has emerged as a promising tool to
improve risk stratification among thyroid nodules placed in these
low-risk indeterminate categories ofThe Bethesda System for
ReportingThyroid Cytopathology (TBSRTC)
• Molecular testing has dual aims in this context
(1) to identify biologically benign nodules that can be
followed clinically rather than surgically and
(2), for nodules that warrant resection, to help guide the
extent of initial surgery (lobectomy versus total
thyroidectomy)

64.  DNA, microRNA, mRNA, and proteins have all been
investigated as analytes for ancillary testing on thyroid cytology
specimens
 The four molecular tests that are currently offered by
commercial laboratories for cytologically indeterminate thyroid
FNAs are all nucleic acid-based tests –
 Afirma Gene Expression Classifier
 RosettaGX Reveal
 ThyGenX/ThyraMIR
 ThyroSeq

65. Afirma
GEC
RosettaGX
Reveal
ThyGenX/
ThyraMIR
ThyroSeq
Testing
approach
Expression
profiles of
142mRNAs by
DNA
microarray
Expression
profiles
of 24
microRNAs by
qRT-PCR
ThyGenX:
Hotspot
mutations in 5
genes and 3
gene fusions by
targeted NGS
ThyraMIR:
Expression
profiles of 10
microRNAs by
qRT-PCR
Hotspot
mutations in
14 genes and
42 gene fusions
by targeted
NGS
Substrate for
molecular
testing
Fresh cells
collected into
nucleic acid
preservative
Fixed cells on
routine
cytology slides
(direct smear
or LBC)
Fresh cells
collected
into nucleic
acid
preservative
Fresh cells
collected into
nucleic acid
preservative

66. Afirma
GEC
RosettaGX
Reveal
ThyGenX/
ThyraMIR
ThyroSeq
Minimum
quantity of
material
required
for molecular
testing
2 dedicated
FNA passes
1 cytology
slide with
sufficient
cellularity
for cytologic
interpreta
1 dedicated
FNA pass
containing
at least 50 ng
of cellular
material
1–2 drops of
FNA material

67.  BCR-ABL trans. for CML , germline mut in the BRCA1/2 for HBOC.
 Rule out tests are much less common (CEA, CA-125, PSA, HPVDNA)
 In thyroid mutation of BRAF ,RAS and rearrangement of RET/PTC
and PAX8/PPAR-γ are most common.
 Analysis of those abnormatlities are considered as rule-in tests for
thyroid.
 NGS likeThyGenX,ThyroSeq are used for this.
 BRAFV600E mutation and RET-PTC1/3 gene fusions are associated with
near-100% risk for PTC.
 In contrast, RAS mut & PAX8-PPARG fusions have been identified in a broad
spectrum of benign, premalignant, and malignant neoplasms (FA, FC,
NIFTP, encapsulated Fptc ) – may be best considered markers of
neoplasia rather than malignancy per se.

68. • An alternative to the rule in approach of mutation panels for the
evaluation is rule out approach of mRNA expression analysis
exemplified by theAfirma Gene Expression Classifier (GEC).
• Unlike the mutation panels, the GEC utilizes an approach that is
designed to look for the presence of benign mRNA expression
patterns in cytologically indeterminate nodules rather than the
absence of specific mutations.
• Has an advantage over mutation analysis in identifying gene
signatures that reflect whole patterns of pathway activation
resulting from both upstream mutations and environmental
factors rather than alterations in a small number of genes

69.  At present, molecular testing is meant to complement and not
replace clinical judgment, sonographic assessment, and visual
cytopathology interpretation
 AmericanThyroidAssociation (ATA) Recommendation
 For patients with a preference for surgical excision, a
molecular test with high specificity and PPV (AFIRMA
GEC) was recommended
 For patients with a preference of conservative management,
a molecular test with high sensitivity and NPV (Four panel
mutation test) was recommended

71. SALIVARY GLAND

72.  Salivary gland tumors (SGTs) are heterogeneous nature with
more than 40 different types of neoplasms described in the
currentWHO classification scheme
 Therefore, in some cases a specific diagnosis may not be
rendered based on morphology alone,
 Several authors have demonstrated that the use of ancillary
techniques (including molecular testing) can overcome the
morphological limitations and refine the diagnostic practice of
salivary gland cytology
 FISH and PCR based assays can be done using freshly prepared
FNA smears, CYTOSPIN preparations, cell blocks

73. Translocations and gene fusions in salivary gland
lesions
SGT Translocations Genes involved Prevalence
Pleomorphic
adenoma
t(3;8)(p21;q12)
HMGA2
rearrangement,
HMGA2 and
MDM2
amplification
PLAG1,
CTNNB1,and
LIFR
HMGA2,
MDM2,and
60%
Mucoepidermoid
carcinoma
t(11;19)(q21–22;p13)
t(6;22)(p21;q12)
MAML2-CRTC1
gene fusion
EWSR1-POU5F1
60–75%
Secretory carcinoma t(12;15)(p13;q25) ETV6-NTRK3
gene fusion
90-100 %
Polymorphous (low-
grad) adenocarcinoma
PRKD1 mutation
PRKD gene family
rearrangements
PRKD1
PRKD1,PRKD2,
PRKD3,ARID1A,or
DDX3X
70%
Cribriform
adenocarcinoma of
minor salivary gland
PRKD
rearrangement
PRKD1,2,3,
ARID1A
80%

74. a, b, low- and
high-power, air-
dried smears
stained with Diff-
Quik® stain.
The alcohol-fixed
on-site smears
stained with
Papanicolaou stain
highlight the
delicate cytoplasm
and nuclear
pleomorphism
(low and high
power, d, e).
A case of fine-needle aspiration
diagnosed as secretory carcinoma
(previously known as mammary
analogue secretorycarcinoma)

75. The break-apart fluorescence
in situ hybridization to
evaluate for disruption of
ETV6 gene shows ETV6
rearrangement (c, f low and
high power)

76.  The integration of molecular diagnostic assays in cytopathology has
added a genomic dimension to the world of diagnostic
cytopathology.
 The variety and versatility of cytology specimen preparations
provide multiple options for performingmolecular assays.
 Novel applications of cytology specimens for molecular diagnostic
assays have redefined and expanded the role of cytopathology in
patient care
 Laboratories must evolve with the changing landscapes of
molecular medicine, embrace new technological advancements,
and optimize these methods into routine cytopathology practice.

77. Newly approved

78. • HPVTesting for Head and Neck Cancers
– HPV-positive OPSqCC have a better prognosis and outcome
than conventional head and neck SqCC.
– p16 IHC is a commonly used surrogate marker for hrHPV
detection
– However criteria for detecting hrHPV using p16 in cytology
specimens is not well defined
– CAP guidelines recommend hrHPV testing on all FNA
specimens of known or suspected OPSqCC when hrHPV
status has not previously been established and for metastatic
SqCC of unknown primary
– RNA / DNA in situ Hybridizations , HPV PCR, liquid-phase
test like Cervista assay can be used

79. Yet to be approved . .

80. • Molecular Diagnostics in Pancreatic and Biliary Cytology (PBC)
– Molecular testing has limited diagnostic utility in PBC but can be
useful in certain settings, particularly for the diagnosis of pancreatic
cyst.
– KRAS/GNAS mutations highly sensitive and specific for the detection
of neoplastic mucinous cysts
– TP53 mutation,loss of SMAD4/DPC4,or loss of p16 detected in pancreatic
cyst fluid supports a high-risk cyst.
– Addition of FISH (UroVysion probe set, cholangiocarcinoma-
/PDAC-specific FISH probe set) and/or NGS to cytology may
improve the diagnostic sensitivity of PBC

81. THANK YOU

82. QUESTIONS
• In UroVysion when all probes show 4 signals,
shall we consider it as positive for
malignancy?

83. • These cells may represent malignancy;
however,
– they may also represent a dividing urothelial cell,
which may be 2N.
– Tetrasomic cells are found more frequently in the
upper urothelial tract and should be interpreted
with caution.
– Once 4 abnormal cells are found, some
laboratories may count 100 consecutive urothelial
cells and provide a percentage of abnormal cells.

84. What is Hybrid Capture ?