Molecular Techniques
L M D allow s the iso la tio n o f single cells o r sm all groups o f cells
o u t o f ro u tin e ly processed cytologic specimens. Today, several
com m ercial systems are available.6 T he y have in c om m o n th a t
an op erator selects the cells o f interest o n a screen. The laser
beam cuts the cells th a t are then placed in a m icrofuge tube,
w here extraction procedure fo r subsequent m olec ular analysis
can be done directly. T he A rcturus system (N a tio n a l Cancer
In s titu te and A rcturus Engineering, M o u n ta in View, C A ), w h ic h
is referred to as laser capture m icrodissection, is based o n local
F ig . 3 6 .2
Screen capture of image analysis system with images of individual
cells for
in te r a c tiv e a n a ly s is
(upper right; multitarget fluorescence in situ
hybridization of bladder washing). Individual cells can be relocated for review
at the microscope due to defined coordinates on the automated stage.
heating o f the cells b y an infrared laser source. T his makes the
cells adhere to the surface o f a cap th rou g h a m e ltin g process
between the cells and a transfer film . The L M D system b y Palm
M icrolaser Technologies uses an u ltra v io le t A laser th rou g h an
inverted microscope. The dissected cancer cells are retrieved in to
the cap b y laser pressure catapulting (Fig. 3 6.5 ) . L M D is p articu-
la rly im p o rta n t in diagnostic cytology, in w h ic h tu m o r cells are
often rare and tig h tly adm ixed w ith b enign cells. C urrently, one
o f the im p o rta n t ro u tin e applications is the iso la tio n o f cells
fro m non-sm all-cell lun g cancer (N SC LC ) fo r epiderm al g row th
factor receptor (EG FR) sequence analysis. In cases o f unselected
sam p ling o f cells fro m a resp iratory cytologic specim en, the
m ore ab und ant n o rm a l D N A could d ilu te and obscure m utated
tu m o r D N A .
Promoter M├ęthylation Analysis
The m e th y la tio n o f cytosine to 5-m ethylcytosine in CpG d in u -
cleotides (w ith
ind ica ting th e phosphate backbone th a t con-
nects th e cytosine and guanine nucleotides) represents a m a in
m echanism o f epigenetic m o d ifica tio n s in h um an s.7,8 The term
e p ig e n e tic s
describes changes in gene fu n c tio n th a t are m ito ti-
cally a n d /o r m e io tic a lly h eritab le and th a t do n o t e nta il a
change in D N A sequence. In th e b roader sense, epigenetics
refers to nuclear inheritance th a t is n o t based on differences in
D N A sequence.
Changes in D N A m e th y la tio n patterns occur in a develop-
m ental stage- and tissue-specific m an ne r as w e ll as in aging and
tum origenesis. M ost notably, ina ctivatio n o f tu m o r suppressor
genes is associated w ith h yp e rm e th yla tio n o f C G -rich stretches
term ed
C p G is la n d s
th a t m ap to the p ro m o te r region o f these
genes (Fig. 3 6.6) . It has becom e increasingly clear th a t aberrant
p ro m o te r m e th y la tio n has a central ro le in tum origenesis. It is
detectable in all phases o f the m ultistep carcinogenesis, and
T a b le 3 6 .2
Selected Fluorescence in situ Hybridization Applications in Cytopathologya
M a te ria l
C h ro m o s o m e s , lo c i, g e n e s
A b e rra tio n
D ia g n o s tic a n d c lin ic a l s ig n ific a n c e
Urine/bladder washing
3, 7, 17, 9p21
Aneusomy, 9p21 deletion
Improved sensitivity, elucidation of equivocal
cytology, prediction of risk of recurrence
Predicts favorable response to chemotherapy,
better prognosis
Non-small-cell lung cancer
EGFR (7p12)
"High polysomy'/amplification
Predicts response to therapy with tyrosine
kinase inhibitors (erlotinib and gefitinib)
Breast cancer
HER2 (17q21)
Predicts response to trastuzumab
TOP2A (17q21)
Predicts response to anthracycline-based
ESR1 (6q25)
Predicts response to tamoxifen
Malignant mesothelioma
Distinction between benign and malignant
mesothelial cells
Lung cancer
6, 5p15, 7p12 (EGFR),
and 8q24 (MYC)
Elucidation of equivocal cytologic findings
Cervix uteri (Papanicolaou smear)
Human papillomavirus
Episomal or integrated
May be useful for analysis of atypical squamous
TERC (3q21), MYC (8q24)
Increased copy number
cells of undetermined significance and predict
risk of progression in dysplasia (low- and high-
grade squamous intraepithelial lesion)
aHematolymphatic tumors and sarcomas not considered.
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