36
Molecular Techniques
load q u a n tific a tio n sem iq uantitative. Som e cross-reactivity o f
the H C 2 probes has been reported w ith H P V types n o t included
in the probe m ix, inc lu d in g som e lo w -ris k types,17 fo r exam ple
H P V 11, 53, 61, 66, 67, 70, 71, and 81. T he use o f the H C 2 assay
in the detection o f H P V as a standalone test o r in com parison
w ith PCR-based assays is reviewed elsewhere in th is chapter.
Applications in Cytology
Improved Diagnosis and Classification of Cancer
Urinary Cytology
M u ltita rg e t FISH im proves th e se n sitivity o f cytology fo r the
detection o f u ro th e lia l neoplasia at a h ig h specificity.18-21 T he
U ro V ysion assay (U roV ysion, A b b o tt M olec ular Inc., Des Plaines,
IL) contains centrom eric probes fo r the chrom osom es 3, 7, and
17 and a locus-specific id e n tifie r fo r the region at 9p21. Polyso-
m ies o f the chrom osom es 3, 7, and 17 are fre q u e n tly observed
in b ladder cancer. Losses o f parts o r all o f chrom osom e 9 are
also c om m on, as th ey arise early in the developm ent o f b o th
p a p illa ry and fla t u ro th e lia l lesions.22 Subsequent progression
is associated w ith c hrom osom al in s ta b ility leading to m u ltip le
c hrom osom al aberrations (Fig. 3 6.9) . M u ltita rg e t FISH has a
se n sitivity o f 9 0 -1 0 0 % fo r the detection o f invasive bladder can-
cer (p T1-4) and a specificity o f over 95% .23 In the c lin ic a lly far
less dangerous category o f low -grade n oninvasive bladder can-
cer (pTa, grade 1 -2 ), FISH increases the se n sitivity o f cytology
fro m 25% to 6 0 -7 5 % . In the setting o f surveillance, FISH can
be used to better estim ate the risk o f recurrence. I f confirm ed
in prospective trials, th is m ig h t a llo w the schedules o f c on trol
cystoscopies to be ta ilore d according to the in d ivid u a l ris k and
u ltim a te ly reduce the n um b er o f cystoscopies in patients w ith
a lo w -risk p rofile.20,21 In d a ily practice, FISH is m ost useful fo r
e lucid ation o f equivocal cytology.24 Equivocal find ing s are par-
tic u la rly a p rob lem after bacillus C a lm e tte -G u e rin tre atm e nt o f
carcinom a in situ, in w h ic h even reactive changes can appear
m ost w orrisom e. W e also do n o t hesitate to apply m ultitarg e t
FISH in d iffic u lt cases o f upper u rin a ry tract cytology. W h e n
an experienced cytologist diagnoses an u ne q uivoc ally positive
cytology, however, FISH is unnecessary, since cells o f tru ly h ig h -
grade u ro th e lia l carcinom a always e xh ib it c hrom osom al aber-
rations.
O ne m ust consider th a t c hrom osom al a b norm alities are n o t
to ta lly restricted to m alig nancy b u t m ay rarely occur in benign
cells. It is w e ll k n o w n th a t te tra p lo id y w ith a balanced duplica-
tio n o f the w h o le genom e can prevail in non-neop lastic condi-
tio n s o f the bladder.25 In contrast, unbalanced num eric changes
o f one o r m ore chrom osom es o r loss o f 9p21 is v irtu a lly specific
fo r neoplasia in b ladder cytology. N otably, pelvic irra d ia tio n
(e.g. o f prostate cancer) o fte n leads to perm anent c hrom o -
som al aberrations, em phasizing the need fo r appropriate c lin i-
cal in fo rm a tio n and consideration o f the typical p o stirrad ia tio n
cytom orp holog ic changes. Despite the rad iation-ind uced chro-
m osom a l aberrations, the risk o f b ladder cancer after irra d ia tio n
fo r prostate cancer is n o t clearly increased.26 Therefore a positive
FISH result in a p atient w ith a h is to ry o f pelvic irra d ia tio n (e.g.
o f prostate cancer) does n o t prove cancer.
The beauty o f FISH lies in the fact th a t chrom osom al genes can
be analyzed directly in the cells o f diagnostic cytologic specimens.
This physical fusion o f m orp h olog y and m olecular genetics has a
great educational effect and helps to sensitize the cytopathologists
F ig . 3 6 .9 Cell o f u r o th e lia l c a rc in o m a w it h m u lt ip le c h ro m o s o m a l
a b e r r a tio n s . Increased c o p y n u m b e rs o f ch ro m o so m e s 3 (Spectrum R ed),
7 (S pectrum G reen), and 17 (Aqua). N ote th a t th e n u m b e r o f 9p21
(S pectrum G old) is lo w e r th a n th e n u m b e r o f th e o th e r signals (3 versus 5 -6 ),
in d ica tin g relative loss o f 9p21.
to subtle m orp holog ic details. In o the r words, the m ore diagnos-
tic feedback b y FISH yo u get, the less you need it.
M ic rosatellite analysis fo r detection o f L O H is another, yet
non-cell-based, m eth od to detect chrom osom al changes in u ri-
nary specimens. T he perform ance o f m icrosatellite analysis in
u rin e has recently been im p roved b y analyzing m u ltip le m ic ro -
satellite m arkers at a tim e and b y d efining m arker-specific thresh-
olds. These technical im p rovem ents led to h ig h ly im p roved
se n sitivity o f up to 72% fo r grade 1 -2 tu m o rs and 96% fo r grade
3 tum ors, at a h ig h specificity o f 8 5-10 0 % . This compares w e ll
w ith the results ob tained b y m u ltita rg e t FISH. Therefore m ic ro -
satellite m arker analysis m ay becom e a PCR-based alternative
to FISH fo r im p roved diagnosis o f bladder cancer in voided
u rin e .27,28 However, the need fo r b lo o d leukocytes fo r n o rm a l
c on trol D N A rem ains a disadvantage o f m icrosatellite analysis
in the urine. Since the m a jo rity o f urines th a t we see in o u r lab o -
ra to ry are cytolog ically unsuspicious and devoid o f tu m o r cells,
there w o u ld be m an y unnecessary b lo o d w ithdraw als.
P ro m o te r h yp e rm e th yla tio n
o f tu m o r suppressor genes
is c om m o n in bladder cancer, and M SP specific to panels o f
selected tu m o r suppressor genes can be used d iag nostically in
void ed u rin e .29 The sen sitivity o f M SP is as h ig h as 8 2 -8 7 % , and
it has up to 100% specificity.29,30 G iven these p rom ising results,
com m ercial assays w ill prob ab ly soon becom e available fo r
p ro m o te r m e th y la tio n analysis in urine.
M alignant Mesothelioma
M esothelial cells can be d ifferentiated fro m carcinom a cells in
m ost cases thanks to w ell-established im m unocytochem ical
m arker panels. The d istin c tio n between reactive m esothelial
changes rem ains d iffic u lt, however, and m an y clinicians are reluc-
ta n t to accept a d e fin itive diagnosis o f m a lig n a n t m esothe liom a
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