THE ROLE OF CYTOLOGY AND SMALL BIOPSIES

IN LUNG CANCER MANAGEMENT

ETTY HARY KUSUMASTUTI

• Lung cancer is one of the most common
causes of cancer-related death worldwide
• 2004 WHO classification recommended that
small specimens should be categorized into:
• NSCLC
• SCLC
 Different management guidelines

• 2011 IASLC/ ATS/ERS suggest of cytology or small

biopsies diagnosed morphologic feature of
– Adenocarcinoma or Squamous cell carcinoma, rather
than NSCLC
– If there is no definitive feature  Immunostains
(NSCLC favor ADC or NSCLC favor SCC)

• Endorse by the 2015 WHO of Lung Cancer

• Tissue samples (small biopsies and cytology) are no
longer managed for diagnosis alone  to potential
targeted therapy.

• Approximately 70% of lung cancers are

inoperable/ advanced stage  molecular targeted
therapies that require molecular testing are primarily
administered to patients with advanced NSCLC.

• Strategic tissue management for ancillary analyses is

critical.
MODALITIES OF TUMOR SAMPLING IN LUNG CANCER

Rodrigiuez EF, Journal of American Society of Cytology (2016)

CORE NEEDLE BIOPSY WITH CT SCAN GUIDED
CORE NEEDLE BIOPSY DENGAN TUNTUNAN ULTRASOUND
 CELL BLOCK

• Some laboratories recommend the routine

preparation of cell blocks for paraffin
embedding of FNB samples  relatively time
consuming and costly compared to routine
smears.
• Cell blocks may give a better idea of tissue
architecture and allow multiple sections for
panels of immune markers with controls.
Problem of cell blocks:
Low-cellularity is the main cause of unsatisfactory
cellblock

Many factors may influence the suboptimal results:

• Operator experience
• Amount of sample
• Location
• Type of lesion
• Variability of CB processing
PROTOCOL FOR PLASMA-THROMBINE METHODE FOR
CELL BLOCK PREPARATION

Shidham VB, 2007

PROTOCOL FOR PREPARATION CELL BLOCKS WITH HISTOGEL

Sidham V.B., 2007

PREPARATION OF CELL BUTTON
• A drop of thick, creamy material obtainable from such tissues using
a needle is gently expelled onto a glass slide.
• After a few seconds to allow the drop to adhere to the slide, the
slide is carefully immersed in 90% ethanol.
• The sample remains stuck to the slide as a drop (‘button’).
• After fixation, the ‘button’ is gently detached with a scalpel blade
and processed like a small biopsy.

Orell S, 2012
 DIAGNOSTIC MORPHOLOGIC EVALUATION OF LUNG
CANCER IN CYTOLOGY DAN SMALL BIOPSIES

• Small biopsies and cytology specimens  NSCLC

should be further classified into a more specific type,
such as adenocarcinoma or squamous cell
carcinoma, whenever possible.

• The term NSCLC-NOS should be used as little as

possible, and only when a more specific diagnosis is
not possible.
• The terms adenocarcinoma in situ (AIS) and
minimally invasive adenocarcinoma should
not be used for diagnosis of small biopsies or
cytology specimens.

• The term large cell carcinoma should not be

used for diagnosis in small biopsy or cytology
specimens and should be restricted to
resection specimens where the tumour is
thoroughly sampled to exclude a
differentiated component.
• Adenosquamous carcinoma is a carcinoma
showing components of both SCC and ADC,
with each components constituting at least
10% of the tumour.
 Definitive diagnosis requires a resection
specimen, although it may be suggested based
on findings in small biopsies, cytology or
excisional biopsies.
• When a diagnosis is made in a small biopsy or
cytology specimen in conjunction with special
studies, it should be clarified whether the
diagnosis was established based on light
microscopy alone or if special stains were
required.

• When paired cytology and biopsy specimens

exist, they should be reviewed together to
achieve the most specific and concordant
diagnosis.
• Judicious use of immunohistochemistry and or
mucin staining is now recommended (when
available) for all cases of NSCLC that can not
be classified as ADC or SCC base on
morphologic alone.

• Ancillary techniques are not always necessary.

Diagnosis small biopsies or cytology
specimens based on morphology alone  50-
70% cases
 Immunostaining in Lung Cancer

IMMUNOSTAIN POSITIVITY IN NSCLC EXPRESSION

TTF-1 80% of lung Nuclear stain
Adenocarcinoma
Napsin A 80% of lung Cytoplasmic stain
Adenocarcinoma

P 63 97% of Squamous Cell Nuclear stain

Carcinoma
P 40 97% of Squamous Cell Nuclear stain
Carcinoma
• Neuroendocrine immunohistochemical
markers should be performed only if cases
where there is suspected neuroendocrine
morphology  CD56, chromogranin, and/or
synaptophysin
• The current WHO guidelines recommend the use of
limited immunostains  TTF-1 & p40
when needed for subclassification in order to save
material for molecular testings.

• Immunostains are performed in order to make a

diagnosis:
– NSCLC, favor adenocarcinoma
– NSCLC, favor squamous cell carcinoma

• The term NSCLC-NOS should be used as little as

possible, and only when a more specific diagnosis is
not possible
 Specimen for Immunostaining
• Tumor tissue in paraffin block
• Cell block
• Unstain direct smear slides
• Slides that are either air-dried or alcohol fixed
 The slides are fixed in formalin for 30-60
minutes  antigen retreival 
Immunostaining
ALGORITHM FOR THE WORK-UP OF NON RESECTION LUNG CANCER SPECIMEN
The pathology report should be including several important
components:

• A pathological or cytopathological diagnosis according

to the IASLC/ATS/ERS classification
• A report of immunohistochemical and/or mucin stains
• A comment about the differential diagnosis (if
appropriate)
• A comment stating that material has been submitted
for molecular testing (if appropriate), specifying which
block or slide is optimal for testing.
MOLECULAR PATHPLOGY OF LUNG CANCER

Recent advances in the field of molecular-based cancer

biology revealed that approximately 60% of adenocarcinomas
and 20% of squamous cell carcinomas have an identified gene
signature, and a finding that has led to the successful
development and approval of targeted therapies
 MOLECULAR ALTERATION IN LUNG CANCER
GENE/PATHWAY MAIN MECHANISM TESTING
EGFR Exon 18 PCR
Exon 19 del
Exon 21 point mutation
Exon 20 (resistance mutation)
KRAS Single amino acid substitution in PCR
codon 12,13 or 61
ALK EML4-ALK fusion gene FISH
IHC
ROS1 ROS1 rearrangement FISH
IHC
KIF5B-RET KIF5B-RET rearrangement IHC

PI3K/AKT/mTOR PI3K mutation PCR

EGFR encodes a transmembrane tyrosine kinase
with an extracellular ligand-binding domain and
an intracellular tyrosine kinase domain.
SPECIMEN FOR MOLECULAR TESTING

The ideal specimens for molecular testing are tumour tissues

obtained fresh and then immediately frozen.

• Formalin-fixed paraffin-embedded block

 specimens should be fixed with buffered formalin (for 6-
24 hours) immediately after tissue acquisition

• Cytologic smear using alcohol-fixed Papanicolaou or air

dried Diff quick stain

Certain fixatives are not recommended for molecular testing:

Bouin solution, B5, Zenker, acidic fixative, decalcifiying
agent
• The minimal number of tumor cells needed
for mutation detection:
200-400 cells

FISH assays require a minimum of 50-100

neoplastic nuclei.
• Next-Generation Sequencing  newer sequencing
technologies that require smaller amount of input DNA
to direct several different molecular alterations
THANK YOU