Copy Number Alterations of DNA Mismatch Repair Genes as Novel Prognostic Markers in Localised Intermediate-risk Prostate Cancer Melvin ChuaA, Emilie LalondeB,C, Osman MahamudA,B, Alejandro BerlinA, Alan Dal PraA, Michèle OrainF, Valerie PicardF, Helene HovingtonF, Alain BergeronF, Yves FradetF, Bernard TêtuF, Alice MengA, Junyan ZhangA, Gaetano ZafaranaA, Julie LivingstoneC, Melania PintilieA, Theodorus van der KwastD, Michael FraserA, Paul BoutrosB,C,E, Robert Glen BristowA,B; CPC-GENE APrincess
Margaret Cancer Centre; BDepartment of Medical Biophysics, University of Toronto; CInformatics & Biocomputing Program, Ontario Institute for Cancer Research; DUniversity Health Network, University of Toronto; Toronto, ON; EDepartment of Pharmacology & Toxicology, University of Toronto; all from Toronto, ON, CA. FUniversite Laval, Quebec city, QC, Canada.
• DNA damage response (DDR) pathways have been implicated in progression of prostate cancer [Mateo, 2015]. • We interrogated if copy number alteration (CNA) of genes involved in DNA Mismatch Repair (MMR) pathway, among others involved in the DDR [Mahamud et al., F11, So et al., C9] are prognostic determinants of biochemical relapses in localised prostate cancer.
Hypotheses: Elevated CNA of genes involved in MMR predicts for biochemical relapse in localised prostate cancer following radical prostatectomy or radiotherapy. We further hypothesise that this molecular profile may be more predictive of treatment failures post-radiotherapy than postprostatectomy.
RESULTS
RESULTS
BACKGROUND & AIM • Summary genomic characteristic like overall copy number burden has been suggested as a prognostic biomarker for biochemical and metastatic relapses in localised prostate cancer [Lalonde, 2014, Hieronymus, 2014].
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Biochemical RFR stratification in low- and intermediate-risk diseasebased on CNA of MMR pathway
Clinical characteristics of study cohort Summary statistics
Clinical factors
MSH2/6/3-deleted
Whole cohort
Radiotherapy
Prostatectomy
(N = 358)
(N = 136)
(N = 222)
Age (years)
Median (range)
65.6 (42.1-82.6)
71.4 (55.4-82.6)
61.8 (42.1-77.1)
T-category
T1/2a
285 (79.6%)
108 (79.4%)
177 (79.7%)
T2b/c
73 (20.4%)
28 (20.6%)
45 (20.3%)
6
61 (17.0%)
34 (25.0%)
27 (12.2%)
7
297(83.0%)
102 (75.0%)
195 (87.8%)
3+3
61 (17.0%)
34 (25.0%)
27 (12.2%)
3+4
223 (62.3%)
71 (52.2%)
152 (68.4%)
4+3
74 (20.7%)
31 (22.8%)
43 (19.4%)
PSA (ng/ml)
median (range)
7.2 (0.7-19.9)
7.9 (0.7-19.9)
6.7 (1.7-19.5)
PGA
median (range)
4.9 (0-39.2)
6.1 (0-39.2)
4.4 (0-31.5)
Gleason’s score Gleason’s pattern
Whole cohort
CNA of MSH2, MSH3, MSH6, MLH1, PMS2 in study cohort of NCCN-defined low- and intermediate-risk prostate cancers Radiotherapy
MLH1/PMS2-gain
Prostatectomy
Whole cohort
• When analysed according to gene subsets with specific functional roles in the MMR pathway, MSH2/6/3 allelic deletion was associated with bRFR on UV analysis for the radiotherapy cohort, albeit this failed to reach statistical significance.
Prostatectomy
• Conversely, MLH1/PMS2 allelic gain was associated with bRFR on UV analysis for the prostatectomy cohort, but not for the radiotherapy.cohort.
Radiotherapy
METHODS Patients
MV analyses, including clinical predictors for bRFR post-treatment in low- and intermediate-risk disease
• 358 patients with pathologically confirmed NCCN-defined low- and intermediate-risk prostate cancers; N = 136, Image-guided radiotherapy (Toronto) and N = 222, Radical prostatectomy (Toronto and Quebec)
Multivariable analyses Whole cohort
• All patients were hormone naive at the time of definitive treatment.
p
HR (95% CI)
p
HR (95% CI)
p
T2b/c vs. T1/2a
1.35(0.86-2.11)
0.19
1.11(0.49-2.53)
0.79
1.48(0.86-2.55)
0.15
≤7.2 vs. >7.2
1.5(1-2.24)
0.047
2.52(1.08-5.86)
0.032
1.39(0.86-2.24)
0.17
1.52(0.96-2.38)
0.072
1.35(0.62-2.95)
0.45
1.82(1.03-3.21)
0.037
MSH2/6/3-deleted
1.77(1-3.13)
0.049
2.78(1.06-7.26)
0.037
1.48(0.73-3.01)
0.28
MLH1/PMS2-gain
1.38(0.79-2.39)
0.255
0.86(0.36-2.25)
0.75
2.19(1.12-4.31)
0.023
• Frequency of CNA of PMS2 (10.3%, N = 37); MSH3 (6.1%, N = 22), MLH1 (3.6%, N = 13), MSH2/6 (3.6%, N = 11)
Clinical factors
• Tissue processing For radiotherapy cohort, a single ultrasound-guided needle biopsy was obtained prior to the start of therapy, as previously described [Lalonde, 2014]. Fresh-frozen prostatectomy specimens were obtained from the University Health Network Pathology BioBank or from the Genito-Urinary BioBank of the Centre Hospitalier Universitaire de Québec (CHUQ). Tumors (with >70% cellularity) were marked on H&E-stained 4 μm sections, macrodissected, and DNA was extracted using phenol:chloroform method.
• Distinct patterns of allelic gains for MLH1/PMS2; losses in all but 1 case, for MSH2/6/3
T-category
Biochemical RFR stratification in low- and intermediate-risk diseasebased on CNA of MMR pathway
PSA (ng/ml)
MSH2/6/3-deleted or MLH1/PMS2 gain
MMR-altered
Whole cohort
Prostatectomy
•
. Statistical considerations • Primary clinical end-point – Biochemical relapse-free rate (bRFR); measured from time of starting treatment to event
• Multivariate (MV) Cox models that included clinical T-category and baseline PSA were used to estimate adjusted hazard ratios.
Genes
Correlation between CNA, SNV counts, and mRNA abundance for the cohort CNA, SNV, mRNA for the genes, not limited to MMR, involved in the DDR [Mahamud et al., F11, So et al., C9]
• For the analysis of MMR pathway, we focused on the following genes: MSH2, MSH3, MSH6, MLH1, PMS2.
• Event-time distributions were estimated using the Kaplan-Meier method, and compared using the Wald’s test.
Prostatectomy
HR(95% CI)
Prognostic determinants for bRFR
Tissue processing and CNA profiling
• CNA profiling SNP microarrays were performed using 200 ng of DNA by Affymetrix OncoScan FFPE Express 3.0. Analysis of Affymetrix OncoScan FFPE Express 3.0 SNP probe assays was performed using .OSCHP files generated by OncoScan Console 1.1 using a custom reference (prostate and thyroid cancers, and hapmap cell lines). BioDiscovery’s Nexus ExpressTM for OncoScan 3 Software was used to call copy number aberrations using the SNP-FASST2 algorithm with default parameters. Gene level copy number aberrations for each patient were identified by overlapping CN segments with RefGene annotation using BEDTools (v2.17.0). Percentage of genome altered (PGA) was calculated for each sample by dividing the number of base-pairs that are involved in a copy number change by the total length of the genome.
Radiotherapy
Radiotherapy
•
Univariable (UV) analysis of gene-level CNA in MMR pathway and association with bRFR for all patients, prostatectomy, and radiotherapy sub-groups Positive association was observed between gene-level CNA in MMR and outcomes following prostatectomy, but not for radiotherapy
•
We observed SNV mutation in TP53 and MSH6 genes only.
•
RB1, MLH1, RAD21, and MSH3 CNA were associated with mutational burden.
•
CNA of RB1, MSH3 and TP53 were correlated with gene expression.
CONCLUSIONS • We observed a distinct pattern in gene-level CNA of the MMR pathway. • MSH2/6/3 losses may be selectively prognostic for RT; and MLH1/PMS2 gains in RadP. • RB1, MLH1, and MSH3 ought to be further investigated as therapeutic targets in prostate cancer.
