“regina elena” national cancer institute michele gallucci anna cianciulli highlights in the...
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“Regina Elena” National Cancer Institute
Michele Gallucci
Anna Cianciulli
Highlights in the management of
Urogenital Cancer
Rome, May 9-10 2008
Biomarkers associated with bladder cancer progression: a potential tool for
individualized therapy?
Multivariables Multivariables prognostic modelprognostic model
Pathological variablesPathological variables
Michele GallucciMichele Gallucci
Biological markersBiological markers
Anna CianciulliAnna Cianciulli
70 % of Ta bladder tumours70 % of Ta bladder tumours
7% of these are G3 tumours (WHO 7% of these are G3 tumours (WHO 1973)1973)
CISCIS 5-10% 5-10%
R. J. SILVESTER TSW UROLOGY (2006) 1 (S2), 15-23
Pathological prognostic factorsPathological prognostic factors
Pathological prognostic Pathological prognostic factorsfactors
5 years follow up 5 years follow up
906 TURB Ta-T1 906 TURB Ta-T1
47% of the patients recurred47% of the patients recurred
9 % progressed to muscle invasive 9 % progressed to muscle invasive diseasedisease
1/3 of patients with 1/3 of patients with CISCIS died died
R. J. SILVESTER TSW UROLOGY (2006) 1 (S2), 15-23
CLINICAL CLINICAL PROGRESSIONPROGRESSION
HYSTOLOGYHYSTOLOGY
UROTHELIUMUROTHELIUM
LAMINA PROPRIALAMINA PROPRIA MEAN DEEP 1.4 mm *MEAN DEEP 1.4 mm *
MUSCULARIS MUCOSAE/MUSCULARIS MUCOSAE/ VESSELSVESSELS
SUBMUCOSASUBMUCOSA
MUSCULARIS PROPRIAMUSCULARIS PROPRIA * * Cheng L. et al., Cancer 1999; 85: 2638 - 47Cheng L. et al., Cancer 1999; 85: 2638 - 47
INVASIVE UROTHELIAL INVASIVE UROTHELIAL CARCINOMACARCINOMA
SUBSTAGING OF T1 DISEASESUBSTAGING OF T1 DISEASE
T1a: T1a: tumors above or into muscularis tumors above or into muscularis mucosaemucosae
(or large vessels).(or large vessels).
T1b: T1b: tumors below muscularis mucosaetumors below muscularis mucosae
(or large vessels). (or large vessels).
T1T1 SUBSTAGINGSUBSTAGING
T1aT1a
MUSCULARIS MUCOSAEMUSCULARIS MUCOSAE
muscularis propriamuscularis propria----------------------------------------------------------------------------------------------------------------------------------------
T1bT1b MUSCULARIS MUCOSAEMUSCULARIS MUCOSAE
muscularis propriamuscularis propria
INVASIVE UROTHELIAL INVASIVE UROTHELIAL CARCINOMACARCINOMA T1a T1a
INVASIVE UROTHELIAL INVASIVE UROTHELIAL CARCINOMACARCINOMA T1b T1b
INVASIVE UROTHELIAL INVASIVE UROTHELIAL CARCINOMACARCINOMA T2 T2
INVASIVE UROTHELIAL INVASIVE UROTHELIAL CARCINOMACARCINOMA
TUMORS INVADING BEYOND TUMORS INVADING BEYOND THE MUSCULARIS MUCOSAE THE MUSCULARIS MUCOSAE
TEND TO BEHAVE LIKE TEND TO BEHAVE LIKE MUSCULARIS PROPRIA MUSCULARIS PROPRIA
INVASIVE (T2) TUMORS.INVASIVE (T2) TUMORS.
Epstein JI et al. in Bladdere biopsy interpretation. Ed. Lippincott Epstein JI et al. in Bladdere biopsy interpretation. Ed. Lippincott Williams & Wilkins, 2004, pag. 95. Williams & Wilkins, 2004, pag. 95.
T1a and T1b as variables predictive of T1a and T1b as variables predictive of disease progressiondisease progression
staging number of cases progression staging number of cases progression raterate
1994 1994 Hasui et al. Hasui et al. T1a T1a 60 60 6.76.7 T1b T1b 28 28 53.553.5 1997 1997 Holmang et al. Holmang et al. T1a T1a 26 26 3636 T1b T1b 38 5838 58
1999 1999 Cheng et al. Cheng et al. T1a T1a 23 1123 11 T1b T1b 21 3221 32
2000 2000 Kondylis et al. Kondylis et al. T1a T1a 32 2232 22 T1b T1b 17 2917 29
2003 2003 Trias et al. Trias et al. T1a T1a 11 911 9 T1b T1b 13 13
30.730.7
2005 2005 Orsola et al. Orsola et al. T1a T1a 25 825 8 T1b T1b 34 3434 34
CARCINOMA IN SITUCARCINOMA IN SITU
CARCINOMA OF THE BLADDERCARCINOMA OF THE BLADDERINVADING THE MUSCULARISINVADING THE MUSCULARISMUCOSAE MUCOSAE (T1b)(T1b) ASSOCIATED ASSOCIATEDWITH WITH CISCIS HAVE AN INCREASED HAVE AN INCREASED RISK OF PROGRESSIONRISK OF PROGRESSION
Bernardini S et al., J Urol 2001; 165: 42 - 46Bernardini S et al., J Urol 2001; 165: 42 - 46
T1 SUBSTAGINGT1 SUBSTAGING
SUBSTAGING OF T1 BLADDER SUBSTAGING OF T1 BLADDER CARCINOMA ACCORDING TO THE CARCINOMA ACCORDING TO THE DEPTH INVASION DEPTH INVASION (MEASURED BY (MEASURED BY
MICROMETER)MICROMETER) PROVIDES PROVIDES SIGNIFICANT PROGNOSTIC SIGNIFICANT PROGNOSTIC
INFORMATION, AND WE INFORMATION, AND WE RECOMMEND THAT IT BE RECOMMEND THAT IT BE
INCORPORATED INTO A FUTURE INCORPORATED INTO A FUTURE TNM SYSTEMTNM SYSTEM
Cheng L et al.: Substaging of T1 bladder carcinoma based on the depth of Cheng L et al.: Substaging of T1 bladder carcinoma based on the depth of invasion as measured by micrometer. Cancer 1999, vol. 15: 1036-1042invasion as measured by micrometer. Cancer 1999, vol. 15: 1036-1042
IF THE BIOPSY ISIF THE BIOPSY IS SUPERFICIAL SUPERFICIAL ANDAND LACKS LACKS
MUSCULARIS PROPRIA, A SECOND MUSCULARIS PROPRIA, A SECOND BIOPSY BIOPSY
SHOULD BE CONSIDEREDSHOULD BE CONSIDERED
Epstein JI et al. in Bladder biopsy interpretation. Ed. Lippincott Epstein JI et al. in Bladder biopsy interpretation. Ed. Lippincott Williams & Wilkins, 2004, pag. 118 Williams & Wilkins, 2004, pag. 118
UROTHELIAL CARCINOMA UROTHELIAL CARCINOMA
HISTOLOGICAL SUBTYPESHISTOLOGICAL SUBTYPES
UrotheliumUrothelium
Tubular variantTubular variant
Nested Nested
MICROPAPILLARY CARCINOMAMICROPAPILLARY CARCINOMA
Ca in situCa in situ
Micropapillary Micropapillary carcinomacarcinoma
‘‘SIGNET RING CELL TYPE’ UROTHELIAL SIGNET RING CELL TYPE’ UROTHELIAL CARCINOMACARCINOMA
Signet ring Signet ring cell cell
SMALL CELL UROTHELIAL CARCINOMASMALL CELL UROTHELIAL CARCINOMA
Early vs. deferred cystectomy
Presence of CIS
Early cystectomy
Deferred cystectomy
Biological markersBiological markers
To identify and monitor those
patients presenting with
superficial tumors who are likely to develop
recurrent or progressive
disease
To provide additional important
informations in patients with muscle invasive carcinomas
concerning their metastatic potential
and response to adjuvant regimens
BIOLOGICAL MARKERSBIOLOGICAL MARKERS
PHENOTYPIC MARKERSPHENOTYPIC MARKERS
• Blood Group Antigens ( ABH antigens )
•Tumor Associated Antigens ( BTA, Nuclear Matrix Proteins)
•Proliferating Antigens ( Ki-67, Proliferating Cell Antigen)
•Cellular Adhesion Molecules (e-cadherin, integrin)
GENOTYPIC MARKERSGENOTYPIC MARKERS
• CYTOGENETIC ALTERATIONS
•MOLECULAR ALTERATIONS OF ONCOGENES
•MOLECULAR ALTERATIONS OF TUMOR SUPPRESSOR GENES
MOLECULAR ALTERATIONS OF ONCOGENES
MOLECULAR ALTERATIONS OF ONCOGENES
• H-ras
•c-myc
•mdm2
•c-erB-2
MOLECULAR ALTERATIONS OF TUMOR SUPPRESSOR GENES
MOLECULAR ALTERATIONS OF TUMOR SUPPRESSOR GENES
Studies on loss of heterozygosity have identified specific allelic deletions in
many bladder cancer. The Rb gene(13q) and the p53 gene (17p) are the best studied tumor suppressor genes.
RETINOBLASTOMA TUMOR SUPPRESSOR GENE
RETINOBLASTOMA TUMOR SUPPRESSOR GENE
“ Inactivation of the Rb gene is thought to be an important step in bladder
cancer progression “ ( Jung I et al, Cancer Control 2000 )
“ Patients with muscle invasive bladder tumors who had lost Rb expression had a
statistically significant shorter 5-year survival ( p=0.001) than those with
normal Rb protein expression “ ( Cordon-
Cardo et al., Scand J Urol Nephrol 2000)
p53 TUMOR SUPPRESSOR GENEp53 TUMOR SUPPRESSOR GENE
“ Increased p53 immunoreactivity has been noted in higher grade and stage bladder cancers and is associated with disease progression, and decreased overall and disease specific survival
“ ( Chatterjee SJ et al, J. J Clin Oncol 2004 )
Richard J. Cote *, Ram H. Datar, 2006
2007, BJU INT
Prospective evaluation of p53 as prognostic marker in T1 transitional cell carcinoma of the bladder
p53 MUTATIONS: CHEMORESISTENCE VERSUS CHEMOSENSITIVITY
Gemcitabine is effective in TCC cell lines independent of p53 status. Urology 2003
Why are the results contradictory?
•One difference is methodological
•p53 affects a remarkable number of cellular processes. Perhaps defects in damage-induced checkpoints enhance chemosensitivity, whereas defects in apoptosis promote drug resistence
• Examination of p53 status alone cannot determine whether the p53 pathway is intact
p21 TUMOR SUPPRESSOR GENEp21 TUMOR SUPPRESSOR GENE
“ Loss of p21 expression was strongly associated with an increased probability of
recurrence and decreased probability of survival in patients with lymphonode negative
organ confined and lymphonode negative extrabladder disease “
( Shariat SF, 2004)
Richard J. Cote *, Ram H. Datar, 2006
Normal Urotheliump53,EGF-R
Low-grade TCC
Ta
High-grade TCC
Ta
High-grade TCC
Superficial
Carcinoma in situ
Stage T1
Stage T2-4
Metastasis
9p-,9q- Rb-,p16, EGF-R, 17p,7
8p-,11p,erbb2
9p-,9q-
Rb-,8p-,3p
N+,M+
p53?
p53?
CHROMOSOME 9CHROMOSOME 9
The genes,p16 and p15, are found in tandem at p21
region.These genes encode members of
a new family of negative cell cycle regulators, which
product function as cyclin-dependent kinase inibitory
molecules
CHROMOSOME 7CHROMOSOME 7
Pycha et al reported that in patients with recurrence ,61% has
trisomy 7.
Other investigators have also shown that
increased copy number of
chromosome 7 is associated with
progression
CHROMOSOME 17CHROMOSOME 17
Cordon-Cardo et al, revealed that deletions of 17p occur only in invasive tumors and are involved in the progression of bladder cancer
Our experienceInterphase cytogenetics of bladder cancer progression ( Cianciulli et al., 2000)Genetic instability in superficial bladder cancer and adjacent mucosa
( Cianciulli et al., 2003)Genetic instability in advanced bladder cancer and adjacent mucosa
( Gallucci et al, 2005)
Adverse genetic prognostic profile identification ( Gallucci et al, 2007)
Interphase cytogenetics of bladder cancer progression: relationship between aneusomy, DNA ploidy pattern, histopathology and clinical outcome
Cianciulli et al.
Int J Clin Lab Res 2000
Interphase cytogenetics of bladder cancer progression: relationship between aneusomy, DNA ploidy pattern, histopathology and clinical outcome
Cianciulli et al.
Int J Clin Lab Res 2000
The aims of this study were:
To gain more insight into genetic changes at the chromosomal level during
different histopathological stages
To compare the sensitivity of FISH and FCM for detection of disturbed DNA
content
To combine pathological variables with genetic markers in order to identify the
phenotypes likely to progress
Aneusomy(meanSD)
Chr.1 Chr.7 Chr.9 Chr.17
Diploid vs
Aneuploid
ns 0.012 ns 0.000
Ta vs T1 T1 vs T2-3
nsns
ns0.032
nsns
ns0.006
G1 vs G2G2 vs G3
nsns
ns0.052
nsns
0.051ns
Cystectomy
No vs Yes ns 0.024 ns 0.014
Recurrence
No vs Yes ns ns ns 0.001
p value; Mann-Whitney test70 patients
p value
Chr.1 Chr.7 Chr.9 Chr.17
Loss ns ns ns ns
Poly ns 0.015 ns 0.051
Aneusomy ns 0.051 ns 0.005
Comparison between superficial (Ta-1) Comparison between superficial (Ta-1) and invasive (T2-3) bladder cancers and invasive (T2-3) bladder cancers
Comparison between superficial (Ta-1) Comparison between superficial (Ta-1) and invasive (T2-3) bladder cancers and invasive (T2-3) bladder cancers
p value Mann-Whitney test
Occurrence and extent of numerical aberrations of chromosomes 7 and 17 may be associated with the evolution of
aggressive growth in bladder cancer and may be a useful indicator for survival. The presence or absence of markers of potential aggressiveness could
determine the course of treatment.
When we compared chromosome 1 and 9 aneusomy with pathological findings and clinical outcome, our results did not reveal statistically significant differences: this alteration is an
early event
UROEPITHELIAL SUPERFICIAL CANCER
MultifocalityRecurrence
A B
MULTIFOCALITY OF UROEPITHELIAL MULTIFOCALITY OF UROEPITHELIAL SUPERFICIAL CANCERSUPERFICIAL CANCER
“Field Cancerization“ Clonal Development
GENETIC INSTABILITY IN SUPERFICIAL BLADDER CANCER AND ADJACENT MUCOSA: AN INTERPHASE
CYTOGENETIC STUDY
GENETIC INSTABILITY IN SUPERFICIAL BLADDER CANCER AND ADJACENT MUCOSA: AN INTERPHASE
CYTOGENETIC STUDY
Cianciulli et al., Human Pathology 2003
The aim of this study was:
To evaluate genetic alterations as a possible reason for multifocality of
uroepithelial superficial cancer
To evaluate genetic alterations as a possible reason for multifocality of
uroepithelial superficial cancer
25 uroepithelial carcinomas
51 tissue samples taken from sites of macroscopically uninvolved urothelium (1cm and
controlateral wall)
CHROMOSOME 1 ANEUSOMY
0
20
40
60
80
BT NPM NDM
ME
AN
% SD
% MEAN
CHROMOSOME 7 ANEUSOMY
0
20
40
60
80
BT NPM NDM
ME
AN
% SD
% MEAN
CHROMOSOME 9 ANEUSOMY
0
20
40
60
80
100
BT NPM NDM
ME
AN
%
SD
% MEAN
CHROMOSOME 17 ANEUSOMY
0
20
40
60
80
BT NPM NDM
ME
AN
%
SD
% MEAN
BT vs NPM:p<0.050
BT vs NDM: p<0.016
BT vs NPM:p<0.050
BT vs NDM: p<0.006
BT vs NPM:p<0.032
BT vs NDM: p<0.004
BT vs NPM:p<0.032
BT vs NDM: p<0.001
CHROMOSOME 1 ANEUSOMY
0
20
40
60
80
100
BT PPM PDM
ME
AN
% SD
% MEAN
CHROMOSOME 7 ANEUSOMY
0
20
40
60
80
BT PPM PDM
ME
AN
% SD
% MEAN
CHROMOSOME 9 ANEUSOMY
0
20
40
60
80
100
BT PPM PDM
ME
AN
%
SD
% MEAN
CHROMOSOME 17 ANEUSOMY
0
20
40
60
80
100
BT PPM PDM
ME
AN
%
SD
% MEAN
BT vs PPM:p<0.033
BT vs PDM: p<0.042
BT vs PPM:p=ns
BT vs PDM: p=ns
BT vs PPM:p=ns
BT vs PDM: p=ns
BT vs PPM:p<0.041
BT vs PDM: p<0.047
CONCLUSIONS
Our results demonstrate a close genetic relationship between all examined tumors and normal-appearing
mucosa
General genetic instability already present in the entire transitional epithelium at the time
of tumor occurrence is a reason for multifocality
Aneusomy of evaluated chromosomes, especially of chromosomes 7 and 9, may represent an intermediate
biomarker of bladder tumorigenesis and could be potentially useful in identifying patients prone to
metachronous or synchronous
BA C
CASE 3
A: T1 G2Chr.1=50Chr.7=18Chr.9=58Chr.17=40
B: Normal UrotheliumChr.1=5Chr.7=18Chr.9=9Chr.17=20
C: Normal UrotheliumChr.1=6Chr.7=1Chr.9=16Chr.17=16
AB
C
CASE 17
A: TaG2Chr.1=40Chr.7=62Chr.9=6Chr.17=38
B: TaG1Chr.1=26Chr.7=52Chr.9=6Chr.17=3
C: FlogosisChr.1=7Chr.7=30Chr.9=5Chr.17=30
Correlation between mean percentage of aneusomic cells and histological classification (p=0.000,r=0.6)
A
B
C
A
B
C
AB
CASE 18
A: T1 G3Chr.1=98Chr.7=9Chr.9=58Chr.17=95
B: CISChr.1=46Chr.7=48Chr.9=47Chr.17=62
C BA
CASE 24
A: T1G3Chr.1=40Chr.7=50Chr.9=42Chr.17=88
B: TaG3Chr.1=20Chr.7=46Chr.9=30Chr.17=36
C: FlogosisChr.1=50Chr.7=82Chr.9=80Chr.17=64
Correlation between mean percentage of aneusomic cells and histological classification (p=0.000,r=0.6)
AB
ABC
48 tumors + 20 non –malignant tissue samples
•Evaluation of genetic characteristics and differences between malignant and no-malignant urothelium
•Evaluation of chromosomal and gene characteristics by stage and lymphonodal status
•The potential role of a panel of genetic markers in patients with advanced tumors that will accurately predict not only the course but also the response to therapy
HISTOLOGICAL DIAGNOSIS
*14 with concomitant
Ca in situ
N°N° TUMORS*TUMORS* N° Distal N° Distal MucosaeMucosae
66 T1 G3 N0T1 G3 N0
T2a G3 N0 (n=5)T2a G3 N0 (n=5) 33
T2b G3 N0 (n=6)T2b G3 N0 (n=6) 22
1313 T2b G3 N2 (n=1)T2b G3 N2 (n=1) 22
T2b G3 N1 (n=1)T2b G3 N1 (n=1) 22
T3a G3 N0 (n=5)T3a G3 N0 (n=5) 55
T3a G3 N2 (n=3)T3a G3 N2 (n=3) 22
1818 T3b G3 N0 (n=5)T3b G3 N0 (n=5)
T3b G3 N1 (n=2)T3b G3 N1 (n=2)
T3b G3 N2 (n=3)T3b G3 N2 (n=3) 22
T4a G3 N0 (n=3)T4a G3 N0 (n=3) 22
T4a G3 N1 (n=3)T4a G3 N1 (n=3)
1111 T4a G3 N2 (n=3)T4a G3 N2 (n=3)
T4a G3 N3 (n=2)T4a G3 N3 (n=2)
Cianciulli et al.
9p21(p16)
Cr.9
Cr. 13
13q14(RB)
Cr. 17
17p13.1(p53)
Cr.3 Cr.7
17q11.2-q12(HER2)
Table 1
Cianciulli et al.
TUMOR WITH CONCOMITANT CIS
(n=14)
TUMOR WITHOUT CONCOMITANT
CIS(n=34)
Chr.17 polysomy
(p= .042)
HER-2 amplification
(p= .040)
Tumor CIS
versus
TumorCIS
*= median value
% DFS1-yr
*<=18 81,5 18>18 76,2 ns 15
*<=22 86,5 19>22 78 ns 15
*<=22 83,8 20>22 72,2 ns 17
*<=38 83,8 21>38 72,9 ns 16
*<=18 91,9 18>18 68 0,05 18
*<=26 80,2 18>26 73,3 22no 79,4 21yes 73,3 19no 100 10yes 71 24no 78,5 20yes 76,2 10
Chromosome 17 monosomy
RB heterozygous deletion
Paz tot
Chromosome 7 polysomy
Chromosome 9 monosomy
Chromosome 9 aneusomy
Chromosome 3 polysomy
p
p53 homozygous deletion ns
9p21 homozygous deletion
ns
ns
0,02
RB homozygous deletion
Disease -free Survival According to the Analysis of Genetic Markers
TAKE HOME MESSAGES TAKE HOME MESSAGES
Chromosome 3, 7, 17 Chromosome 3, 7, 17 aneusomy and RB1 aneusomy and RB1
heterozygous deletion heterozygous deletion and 9p21 homozygous and 9p21 homozygous
deletion might be deletion might be potentially useful potentially useful
intermediate intermediate biomarkers to detect biomarkers to detect those patients at high those patients at high
risk of progression who risk of progression who may benefit from may benefit from
particular and particular and innovative therapeutic innovative therapeutic
interventionsinterventions
Larger , longterm with Larger , longterm with follow up are needed follow up are needed to assess the validy to assess the validy and clinical relevance and clinical relevance of these genetic of these genetic findingsfindings
Predictors of good prognosis
Predictors of poor prognosis
MCA plot showing the projections of the five identified biological variable ( active information) and death ( passive information)
Prognostic factors based model: the presence of two
variables significantly
impair overall survival!
New genetic information must be incorporated intocurrent schema to gain a better understanding of
tumorigenesis, biologic pathways and ultimate treatment outcomes.
Hopefully, it will soon be possible to use a panel of markers to identify patients at greatest risk for progression: that will significantly
improve the clinical management of cancer.