PART THREE
Special Techniques in Cytology
based o n cytology alone. The m a jo rity o f m a lig n a n t m esothe lio -
mas (> 70% ) show hom ozygous d eletion o f the C D K N 2A gene at
9p21.31,32 C D K N 2A encodes fo r the p16 (IN K 4 a) p rotein, w h ic h
is a negative reg ulator o f cell g row th.31,32 In a sm a lle r fra ctio n o f
m esotheliom as (< 20% ), C D K N 2A is inactivated b y p ro m o te r
m e th y la tio n .33 In a d d itio n to p16 ina ctivatio n b y hom ozygous
d ele tio n o r p ro m o te r m e th yla tio n , num eric c hrom osom al aber-
rations inc lu d in g p o lysom y (m o s tly triso m y) o f chrom osom e 7
are c om m o n in m esothe liom a (> 8 0 % ).31,32 FISH is w e ll suited
fo r jo in t analysis o f b o th 9p21 d ele tio n and chrom osom al p o ly-
som y (Fig. 36.10) and m ay thus a llo w a d e fin itive diagnosis o f
m a lig n a n t m esotheliom a. Analysis o f p ro m o te r m e th y la tio n
o f C D K N 2A m ig h t provide diagnostic in fo rm a tio n in cases
w ith o u t hom ozygous 9p21 deletion.
Lung Cytology
Fluorescence In Situ Hybridization
A m u ltita rg e t FISH assay (LA Vysion, A b b o tt M olec ular Inc.) fo r
the sim ultaneous analysis o f chrom osom e 6 and the 5p15, 7p12
(EG FR gene) and 8q24 (M Y C gene) loc i was designed to im p rove
se n sitivity in lun g cytology.34 This c o m b in a tio n o f FISH probes
revealed a se n sitivity o f 100% w h e n applied to touch prepara-
tio n s fro m NSC LC s.35 I f applied to b ron c hial brush cytology, it
increases the se n sitivity o f cytology fo r the detection o f p erip h-
eral NSC LC fro m 30 to 7 0% .36,37 T his im p roved se n sitivity over
cytology can be explained by a localized cancerization field in
the b ron c hial m ucosa adjacent to the invasive cancer. A lth o u g h
p rom ising , th is se n sitivity is s till n o t satisfactory, in as m uch as
it is at the cost o f a decreased specificity, w ith ab out 10% false
positives. The diagnostic u tility o f th is m u ltita rg e t FISH in b ro n -
chial secretions and in centrally located tu m o rs is questionable.
Its m ost interesting ap p lication lies in the evaluation o f equivo-
cal resp iratory cytology occurring in ap p roxim ately 2% o f all
patients w h o undergo bronchoscopic e valuation.3 I f com bined
w ith autom ated relocation o f the cells in question, th is assay
is a p ow erful to o l to c la rify equivocal lu n g cytology, reaching a
se n sitivity o f 79% and a specificity o f close to 100%. Thus lack
F ig . 3 6 .1 0 Four cells o f m a lig n a n t m e s o th e lio m a w it h in c re a s e d c o p y
n u m b e rs o f c h ro m o s o m e s 3 (S pectrum R ed), 7 (S pectrum G reen), and 17
(Aqua). Loss o f b o th copies o f 9p21 (S pectrum G old), in d ica tin g h o m o zyg o u s
d e le tio n . N ote th e tw o norm a l cells w ith norm a l c o p y n um b e rs (b o tto m ).
o f a negative FISH result in atypical cells is in favor o f reactive
changes, and an expectant strategy w ith clinical controls can be
considered in these patients. In contrast, the presence o f chro-
m osom al aberrations is strong ly in favor o f cancer and w arrants
fu rth e r actions fo r m orp h olog ic p ro o f o f cancer w ith o u t delay.
In a d d itio n to num eric chrom osom e and gene copy num b er
gains, ina ctivatio n o f a n um b er o f genes b y d eletion a n d /o r
m e th yla tio n , inc lu d in g C D K N 2A at 9p21, F H IT at 3p14, hyal-
uronidase-2 (H YA L2) at 3p21, and surfactant p ro te in A (SP-A)
at 10q21, has been show n to be associated w ith sm o kin g h isto ry
and m ay be an early event d uring cancerogenesis.38 FISH analy-
sis fo r deletions o f F H IT and C D K N 2A in sp utum specimens has
som e p rom ise fo r the screening o f h ig h -risk patients.39
Prom oter Methylation
A b errant m e th y la tio n o f various genes inc lu d in g C D K N 2A and
RASSF1A has been suggested as a b io m a rke r fo r im p roved lung
cancer diagnostics as w e ll as fo r lu n g cancer screening, w ith
p rom ising results.40-42 In patients suspected o f having lun g can-
cer, b ron c hial aspirates m ay be valuable fo r m e th y la tio n analy-
sis, whereas lu n g cancer screening relies o n sp utum samples. In
contrast to never sm okers, patients w ith a h is to ry o f heavy sm o k-
ing m ay show aberrant p ro m o te r m e th yla tio n , even th ou g h at a
lo w level. Therefore any m e th y la tio n assay fo r lung cancer diag-
nostics w ill have to introd uce a th resh old characterizing a posi-
tive test result. A t present, c om m ercially available assays are in
developm ent.
Other Applications
O ligodendrogliom a
D eletions o f chrom osom al regions at 1p and 19q predict a favo-
rable response to chem otherap y and im p roved survival in o lig o -
d end rog liom a and high-grade g lio m a .43 These chrom osom al
aberrations are preferably analyzed b y FISH o n cytologic touch
preparations.44
Barrett's Esophagus
Fluorescence in situ h yb rid iz a tio n com bined w ith cytology
m ay becom e useful in the surveillance o f patients w ith Barrett's
esophagus.45 Barrett's esophagus is a m etaplastic c o n d itio n o f
the distal esophagus th a t is associated w ith an increased risk
fo r developing esophageal adenocarcinom a. Therefore patients
w ith Barrett's esophagus need to undergo regular surveillance
fo r early detection o f high-grade dysplasia and adenocarcinom a.
However, the d istin c tio n o f dysplasia fro m reactive changes is
d iffic u lt, b o th by cytology and o n h isto lo g y o f Barrett's esopha-
gus, and suffers fro m hig h interobserver and intraobserver va ri-
ab ility. Instead, the c o m b in a tio n o f FISH probes to 8q24, 9p21,
17q11.2, and 20q13.2 was reported to have a se n sitivity and
specificity o f 84% and 93% , respectively, fo r high-grade dyspla-
sia, and 94% and 93% , respectively, fo r esophageal adenocar-
cinom a.45 I f confirm ed in prospective studies, FISH on brush
cytology could becom e a standard fo r surveillance o f patients
w ith Barrett's esophagus.
Biliary Tract and Pancreas
The diagnosis o f b ilia ry tract adenocarcinom a is a no the r n o to ri-
ously d iffic u lt field due to m orp h olog ic overlap between reactive
and m alig n an t b ilia ry cells. P olysom y and hom ozygous d eletion
o f 9p21 are c om m o n in cholangiocarcinom a, m aking m u ltita rg e t
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