FISH Analysis in Urothelial Cancer Michael Neat, Dr M Mason and Dr A Chandra.

FISH Analysis in FISH Analysis in Urothelial CancerUrothelial Cancer

Michael Neat, Dr M MasonMichael Neat, Dr M Masonand Dr A Chandraand Dr A Chandra

Interphase FISH in Interphase FISH in urothelial carcinomaurothelial carcinoma

Low sensitivity of urine cytology esp in low Low sensitivity of urine cytology esp in low grade lesionsgrade lesions– Need for additional tests for detection and Need for additional tests for detection and

monitoringmonitoring– In conjunction with not in lieu of routine proceduresIn conjunction with not in lieu of routine procedures

The UroVysion FISH assayThe UroVysion FISH assay– First published 2000; 4 loci with best combined First published 2000; 4 loci with best combined

sensitivity from 10 candidates sensitivity from 10 candidates – 2 FDA trials2 FDA trials

2001 FDA approved for detection of recurrence2001 FDA approved for detection of recurrence 20052005

– Pts with haematuriaPts with haematuria– No prev Hx Ca bladderNo prev Hx Ca bladder– Ca bladder histologically Dx in 50/497 (10.2%)Ca bladder histologically Dx in 50/497 (10.2%)– FISH detected 69% of these, cytology 38%FISH detected 69% of these, cytology 38%– When TaG1 tumours excluded; FISH 83%, cytology 50%When TaG1 tumours excluded; FISH 83%, cytology 50%– FDA approval 2005 pts with haematuriaFDA approval 2005 pts with haematuria

Abbott MolecularMix of 4 probes labelled with 4 different fluorochromesUnstained ThinPrep slides

The FISH assayThe FISH assay

Analysis/scoring criteriaAnalysis/scoring criteria Initially select morphologically abnormal Initially select morphologically abnormal

cellscells– Large nuclear size/irregular shapeLarge nuclear size/irregular shape– Patchy DAPI stainPatchy DAPI stain– Cell clusters (non-overlapping)Cell clusters (non-overlapping)– If no morphologically abnormal cells present, If no morphologically abnormal cells present,

scan all cellsscan all cells Minimum analysis of 25 cellsMinimum analysis of 25 cells FISH positive if:FISH positive if:

– ≥≥4 cells showing gain of at least 2 of #3, #7 & 4 cells showing gain of at least 2 of #3, #7 & #17#17

– ≥≥12 cells showing homozygous deletion of 12 cells showing homozygous deletion of p16p16 i.e. no i.e. no p16 p16 signalssignals

Examples of abnormal Examples of abnormal signal patternssignal patterns

Homozygous

deletion of p16

Increased copy no.

of #3, #7 & #17

ICN & homozygous

deletion of p16

Success rateSuccess rate

Analysis successful 58/59 (98%) casesAnalysis successful 58/59 (98%) cases– 1/59 – post treatment, probe hyb failed - ? 1/59 – post treatment, probe hyb failed - ?

DNA degradedDNA degraded– 14/58 (24%) FISH positive14/58 (24%) FISH positive

Highly reproducible assay Highly reproducible assay when when samples adequatesamples adequate– 12/71 (17%) samples received insufficient 12/71 (17%) samples received insufficient

materialmaterial– Caraway Caraway et al et al 65/1006 (6%) insufficient 65/1006 (6%) insufficient

(cytospin)(cytospin)

Performance of the assayPerformance of the assay Halling & KippHalling & Kipp

Eur Ren Genotourinary Dis.Eur Ren Genotourinary Dis. 2006;2:51-542006;2:51-54– Mean sensitivity of FISH cf. Mean sensitivity of FISH cf.

cytology in 12 studiescytology in 12 studies

– Cytology specificity higher Cytology specificity higher than FISH (93% vs. 85%)than FISH (93% vs. 85%)

Stage/Stage/gradegrade

FISH (%)FISH (%) Cytology Cytology (%)(%)

TaTa 6767 2828

TisTis 9797 7373

T1T1 9090 6767

T2-T4T2-T4 9292 7474

Grade 1Grade 1 5050 1818

Grade 2 Grade 2 7575 4545

Grade 3Grade 3 9090 6969

Stage/Stage/gradegrade

FISH (%)FISH (%) Cytology Cytology (%)(%)

AllAll 7272

(69-75)(69-75)4242

(38-45)(38-45)

Excluding Excluding TaTa

8686

(82-89)(82-89)6161

(56-66)(56-66)

HajdinjakHajdinjakUrol Oncol. Urol Oncol. 2008;26:646-6512008;26:646-651– Meta-analysis (2477 Meta-analysis (2477

FISH tests in 14 FISH tests in 14 studies, cytology from studies, cytology from 12)12)

– Cytology specificity Cytology specificity higher than FISH (96% higher than FISH (96% vs. 83%)vs. 83%)

Conflicting dataConflicting data– May May et al. et al. Urology 2007;70(3):449-53Urology 2007;70(3):449-53

Conventional cytology can be better than FISH Conventional cytology can be better than FISH in experienced handsin experienced hands

Sensitivity 71% vs. 53.2%Sensitivity 71% vs. 53.2% Specificity 83% vs. 74%Specificity 83% vs. 74%

– Moonen Moonen et al. et al. Eur UrolEur Urol 2007;51(5):1275-80 2007;51(5):1275-80 No improvement over cytology in detection of No improvement over cytology in detection of

recurrencerecurrence Sensitivity 40.6% vs. 39.1%Sensitivity 40.6% vs. 39.1% Specificity 89.7% vs. 89.7%Specificity 89.7% vs. 89.7%

Clinical applicationsClinical applications Detection of recurrenceDetection of recurrence Gross or microscopic haematuriaGross or microscopic haematuria Anticipatory positive resultsAnticipatory positive results

– FISH can detect tumour before clinically FISH can detect tumour before clinically detectable by cytoscopy or cytologydetectable by cytoscopy or cytology

Helpful for clarifying equivocal cytology in Helpful for clarifying equivocal cytology in patients with equivocal or negative patients with equivocal or negative cytoscopycytoscopy

? detection of non-UC bladder tumours? detection of non-UC bladder tumours– Histological variants detected on FFPE’sHistological variants detected on FFPE’s– ? Exfoliating tumours? Exfoliating tumours

Clinical applications Clinical applications (cont.)(cont.) Follow-up post intravesical therapyFollow-up post intravesical therapy

– BCG-associated inflammation makes cytoscopic & BCG-associated inflammation makes cytoscopic & cytologic interpretation difficultcytologic interpretation difficult

– Savic Savic et alet al 68 pts; NMIBC, post BCG68 pts; NMIBC, post BCG Both positive cytology and positive FISH predict Both positive cytology and positive FISH predict

failure of BCGfailure of BCG FISH superior when cytology non-definitive, i.e. FISH superior when cytology non-definitive, i.e.

equivocal, mild or moderate atypiaequivocal, mild or moderate atypia– Whitson Whitson et alet al

Positive FISH after IVT significant predictor of Positive FISH after IVT significant predictor of recurrence in multivariate analysisrecurrence in multivariate analysis

Detection of upper tract UCDetection of upper tract UCAuthorAuthor FISH (%)FISH (%) Cytology (%)Cytology (%)

Marin-Aguilera Marin-Aguilera et alet al

76.776.7 3636

Akkad Akkad et alet al 87.587.5 6060

DisadvantagesDisadvantages CostCost Technical & interpretive difficultiesTechnical & interpretive difficulties

– TrainingTraining– equipmentequipment

False positivesFalse positives– BK polyoma virus (rare)BK polyoma virus (rare)– TetraploidyTetraploidy

Reactive urothelial cellsReactive urothelial cells Cells in S or G2 phaseCells in S or G2 phase ? Less specific predictor of malignancy? Less specific predictor of malignancy

False negativesFalse negatives– low-grade neoplasms if representative cells are not low-grade neoplasms if representative cells are not

shed into the urine sampleshed into the urine sample– Lack of atypical cells on the slide used for FISHLack of atypical cells on the slide used for FISH

ConclusionsConclusions

Useful adjunctive assay to increase Useful adjunctive assay to increase sensitivity in targeted patient sensitivity in targeted patient populationspopulations

In routine use in many countriesIn routine use in many countries Developing assayDeveloping assay Does earlier detection translate into Does earlier detection translate into

decreased mortality?decreased mortality? Is negative predictive value Is negative predictive value

sufficient to decrease the need for or sufficient to decrease the need for or frequency of cytoscopic follow-up?frequency of cytoscopic follow-up?

Total FISH tests Total FISH tests undertakenundertaken

NegativePositive

43

13

Total tests = 56

(41 patients)

Follow up dataFollow up data

4137

2215

05

1015202530354045

Total

patie

nts

Cysto

scop

y perf

ormed

Hist

ology

No h

istolo

gy

Total number

CorrelationCorrelation

11

12

18

4

4

4

0 5 10 15 20 25

Cyt

olo

gy-

His

tolo

gyC

ytolo

gy-

FIS

H

FIS

H-

His

tolo

gy

MatchMis-match

Sensitivity and Specificity of FISH with Sensitivity and Specificity of FISH with histologyhistology

FISH NEGATIVEFISH NEGATIVE FISH POSITIVEFISH POSITIVE

HISTOLOGY HISTOLOGY NEGATIVENEGATIVE

1414 22

HISTOLOGY HISTOLOGY POSITIVEPOSITIVE

22 44

SENSITIVITYSENSITIVITY 66.7%66.7%

SPECIFICITYSPECIFICITY 87.5%87.5%

Cytology categoriesCytology categories

C1: Unsuitable for diagnosisC1: Unsuitable for diagnosis C2: BenignC2: Benign C3: Atypia, probably reactive C3: Atypia, probably reactive

(expected outcome – 10-15% (expected outcome – 10-15% malignant)malignant)

C4: Atypia, probably malignantC4: Atypia, probably malignant C5: MalignantC5: Malignant

Sensitivity and Specificity of cytology Sensitivity and Specificity of cytology with histology (C3=NEGATIVE)with histology (C3=NEGATIVE)

CYTOLOGY CYTOLOGY NEGATIVE (C 1,2,3)NEGATIVE (C 1,2,3)

CYTOLOGY CYTOLOGY POSITIVE POSITIVE (C4,5)(C4,5)

HISTOLOGY HISTOLOGY NEGATIVENEGATIVE

66 33

HISTOLOGY HISTOLOGY POSITIVEPOSITIVE

11 55

SENSITIVITY 62.5%

SPECIFICITY 85.7%

C3 cytology and FISHC3 cytology and FISH

C3 FISHnegativeC3 FISH positive

4

17

Total tests = 21

Future applicationsFuture applications

Emerging evidence that persistent Emerging evidence that persistent positive FISH following BCG positive FISH following BCG treatment is predictive of stage treatment is predictive of stage progression of bladder cancerprogression of bladder cancer

Cystectomy may be offered to these Cystectomy may be offered to these patients following a course of BCG patients following a course of BCG and positive FISH testand positive FISH test

AcknowledgmentsAcknowledgments

The UroCyt vials used in this study The UroCyt vials used in this study were provided by Hologic. were provided by Hologic.