Comparison of chromosomal and array-based comparative genomic hybridization for the detection of genomic imbalances in primary prostate carcinomas

BACKGROUND: In order to gain new insights into the molecular mechanisms involved in prostate cancer, we performed array-based comparative genomic hybridization (aCGH) on a series of 46 primary prostate carcinomas using a 1 Mbp whole-genome coverage platform. As chromosomal comparative genomic hybridization (cCGH) data was available for these samples, we compared the sensitivity and overall concordance of the two methodologies, and used the combined information to infer the best of three different aCGH scoring approaches. RESULTS: Our data demonstrate that the reliability of aCGH in the analysis of primary prostate carcinomas depends to some extent on the scoring approach used, with the breakpoint estimation method being the most sensitive and reliable. The pattern of copy number changes detected by aCGH was concordant with that of cCGH, but the higher resolution technique detected 2.7 times more aberrations and 15.2% more carcinomas with genomic imbalances. We additionally show that several aberrations were consistently overlooked using cCGH, such as small deletions at 5q, 6q, 12p, and 17p. The latter were validated by fluorescence in situ hybridization targeting TP53, although only one carcinoma harbored a point mutation in this gene. Strikingly, homozygous deletions at 10q23.31, encompassing the PTEN locus, were seen in 58% of the cases with 10q loss. CONCLUSION: We conclude that aCGH can significantly improve the detection of genomic aberrations in cancer cells as compared to previously established whole-genome methodologies, although contamination with normal cells may influence the sensitivity and specificity of some scoring approaches. Our work delineated recurrent copy number changes and revealed novel amplified loci and frequent homozygous deletions in primary prostate carcinomas, which may guide future work aimed at identifying the relevant target genes. In particular, biallelic loss seems to be a frequent mechanism of inactivation of the PTEN gene in prostate carcinogenesis

Prediction of relapse-free survival according to adjuvant chemotherapy and regulator of chromosome condensation 2 (RCC2) expression in colorectal cancer

Background There is a need for improved selection of patients for adjuvant chemotherapy after resection of non-metastatic colorectal cancer (CRC). Regulator of chromosome condensation 2 (RCC2) is a potential prognostic biomarker. We report on the establishment of a robust protocol for RCC2 expression analysis and prognostic tumour biomarker evaluation in patients who did and did not receive adjuvant chemotherapy. Materials and methods RCC2 was analysed in 2916 primary CRCs from the QUASAR2 randomised trial and two single-hospital Norwegian series. A new protocol using fluorescent antibody staining and digital image analysis was optimised. Biomarker value for 5-year relapse-free survival was analysed in relation to tumour stage, adjuvant chemotherapy and the molecular markers microsatellite instability, KRAS/BRAF(V600E)/TP53 mutations and CDX2 expression. Results Low RCC2 expression was scored in 41% of 2696 evaluable samples. Among patients with stage I-III CRC who had not received adjuvant chemotherapy, low RCC2 expression was an independent marker of inferior 5-year relapse-free survival in multivariable Cox models including clinicopathological factors and molecular markers (HR 1.45, 95% CI 1.09 to 1.94, p=0.012, N=521). RCC2 was not prognostic in patients who had received adjuvant chemotherapy, neither in QUASAR2 nor the pooled Norwegian series. The interaction between RCC2 and adjuvant chemotherapy for prediction of patient outcome was significant in stage III, and strongest among patients with microsatellite stable tumours (p(interaction)=0.028). Conclusions Low expression of RCC2 is a biomarker for poor prognosis in patients with stage I-III CRC and seems to be a predictive biomarker for effect of adjuvant chemotherapy.Peer reviewe

MGMT promoter methylation in gliomas-assessment by pyrosequencing and quantitative methylation-specific PCR

Background Methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene promoter is a favorable prognostic factor in glioblastoma patients. However, reported methylation frequencies vary significantly partly due to lack of consensus in the choice of analytical method. Method We examined 35 low- and 99 high-grade gliomas using quantitative methylation specific PCR (qMSP) and pyrosequencing. Gene expression level of MGMT was analyzed by RT-PCR. Results When examined by qMSP, 26% of low-grade and 37% of high-grade gliomas were found to be methylated, whereas 97% of low-grade and 55% of high-grade gliomas were found methylated by pyrosequencing. The average MGMT gene expression level was significantly lower in the group of patients with a methylated promoter independent of method used for methylation detection. Primary glioblastoma patients with a methylated MGMT promoter (as evaluated by both methylation detection methods) had approximately 5 months longer median survival compared to patients with an unmethylated promoter (log-rank test; pyrosequencing P = .02, qMSP P = .06). One third of the analyzed samples had conflicting methylation results when comparing the data from the qMSP and pyrosequencing. The overall survival analysis shows that these patients have an intermediate prognosis between the groups with concordant MGMT promoter methylation results when comparing the two methods. Conclusion In our opinion, MGMT promoter methylation analysis gives sufficient prognostic information to merit its inclusion in the standard management of patients with high-grade gliomas, and in this study pyrosequencing came across as the better analytical method

Biallelic inactivation of TP53 rarely contributes to the development of malignant peripheral nerve sheath tumors

About 10% of the patients with neurofibromatosis type 1 (NF1) develop malignant peripheral nerve sheath tumors (MPNSTs), accounting for half of all MPNST cases. Several nonrandom chromosomal aberrations have been found, but the target genes remain mostly unrecognized. Mutations in the NF1 and TP53 genes have been found in some MPNSTs, and recent data from mouse models support a synergistic effect of these two genes in the development of MPNST. In the present study, we have analyzed 16 MPNSTs, including 11 from patients with NF1 and 5 sporadic cases, for mutations in the coding sequence of the TP53 gene (exons 2-11). We applied denaturing gradient gel electrophoresis and modifications of this technique for analyses of 12 genomic fragments, followed by direct sequencing for identification of the mutated base(s). None of the MPNSTs revealed mutations. The detection of control mutants for each fragment analyzed, the high sensitivity of the technique, the detection of polymorphisms in some samples, and the high content of tumor tissue in the biopsies imply that false negatives are highly unlikely. Although we cannot exclude that deletions including large parts of the gene remain undetected by the mutation analyses, previous comparative genomic hybridization (CGH), cytogenetic banding analysis, and/or loss of heterozygosity studies on 14 of the cases included here had revealed 17p deletions in only three. We thus conclude that TP53 biallelic inactivation is rare in MPNST, and that the potential impact of an altered TP53 pathway on the malignant transformation of a neurofibroma into an MPNST may more frequently occur by changes in other components of that pathway

Genomic and prognostic heterogeneity among RAS/BRAFV600E/TP53 co-mutated resectable colorectal liver metastases

Hepatic resection is potentially curative for patients with colorectal liver metastases, but the treatment benefit varies. KRAS/NRAS (RAS)/TP53 co‐mutations are associated with a poor prognosis after resection, but there is large variation in patient outcome within the mutation groups, and genetic testing is currently not used to evaluate benefit from surgery. We have investigated the potential for improved prognostic stratification by combined biomarker analysis with DNA copy number aberrations (CNAs), and taking tumor heterogeneity into account. We determined the mutation status of RAS, BRAFV600, and TP53 in 441 liver lesions from 171 patients treated by partial hepatectomy for metastatic colorectal cancer. CNAs were profiled in 232 tumors from 67 of the patients. Mutations and high‐level amplifications of cancer‐critical genes, the latter including ERBB2 and EGFR, were predominantly homogeneous within patients. RAS/BRAFV600E and TP53 co‐mutations were associated with a poor patient outcome (hazard ratio, HR, 3.9, 95% confidence interval, CI, 1.3–11.1, P = 0.012) in multivariable analyses with clinicopathological variables. The genome‐wide CNA burden and intrapatient intermetastatic CNA heterogeneity varied within the mutation groups, and the CNA burden had prognostic associations in univariable analysis. Combined prognostic analyses of RAS/BRAFV600E/TP53 mutations and CNAs, either as a high CNA burden or high intermetastatic CNA heterogeneity, identified patients with a particularly poor outcome (co‐mutation/high CNA burden: HR 2.7, 95% CI 1.2–5.9, P = 0.013; co‐mutation/high CNA heterogeneity: HR 2.5, 95% CI 1.1–5.6, P = 0.022). In conclusion, DNA copy number profiling identified genomic and prognostic heterogeneity among patients with resectable colorectal liver metastases with co‐mutated RAS/BRAFV600E/TP53

Exploratory analyses of consensus molecular subtype-dependent associations of TP53 mutations with immunomodulation and prognosis in colorectal cancer

Accumulating evidence suggests immunomodulatory and context-dependent effects of TP53 mutations in cancer. We performed an exploratory analysis of the transcriptional, immunobiological and prognostic associations of TP53 mutations within the gene expression-based consensus molecular subtypes (CMSs) of colorectal cancer (CRC).Materials and methodsIn a single-hospital series of 401 stage I–IV primary CRCs, we sequenced the whole coding region of TP53 and analysed CMS-dependent transcriptional consequences of the mutations by gene expression profiling. Immunomodulatory associations were validated by multiplex, fluorescence-based immunohistochemistry of immune cell markers. Prognostic associations of TP53 mutations were analysed in an aggregated series of 635 patients classified according to CMS, including publicly available data from a French multicentre cohort (GSE39582). mutations were found in 60% of the CRCs. However, gene set enrichment analyses indicated that their transcriptional consequences varied among the CMSs and were most pronounced in CMS1-immune and CMS4-mesenchymal. Subtype specificity was primarily seen as an upregulation of gene sets reflecting cell cycle progression in CMS4 and a downregulation of T cell activity in CMS1. The subtype-dependent immunomodulatory associations were reinforced by significant depletion of several immune cell populations in mutated tumours compared with wild-type (wt) tumours exclusively in CMS1, including cytotoxic lymphocytes (adjusted p value in CMS1=0.002 and CMS2−4>0.9, Microenvironment Cell Populations (MCP)-counter algorithm). This was validated by immunohistochemistry-based quantification of tumour infiltrating CD8+ cells. Within CMS1, the immunomodulatory association of TP53 mutations was strongest among microsatellite stable (MSS) tumours, and this translated into a propensity for metastatic disease and poor prognostic value of the mutations specifically in the CMS1/MSS subtype (both series overall survival: TP53 mutation vs wt: HR 5.52, p=0.028).Integration of TP53 mutation status with the CMS framework in primary CRC suggested subtype-dependent immunobiological associations with prognostic and potentially immunotherapeutic implications, warranting independent validation

Transcriptional and functional consequences of TP53 splice mutations in colorectal cancer

TP53 mutations are common in colorectal cancer (CRC). Most TP53 sequencing studies have been restricted to coding regions, but recent studies have revealed that splice mutations can generate transcript variants with distinct tumorigenic and prognostic properties. Here, we performed unrestricted sequencing of all coding sequences and splice regions of TP53 in a single-hospital series of 401 primary CRCs. TP53 splice mutations were detected in 4% of the cases (N = 16), considerably more frequent than reported in major databases, and they were mutually exclusive to exon mutations. RNA sequencing revealed high-level expression of aberrant transcript variants in the majority of splice mutated tumors (75%). Most variants were predicted to produce truncated TP53 proteins, including one sample expressing the potentially oncogenic and druggable p53ψ isoform. Despite heterogeneous transcript structures, downstream transcriptional profiling revealed that TP53 splice mutations had similar effects on TP53 target gene expression and pathway activity as exonic mutations. Intriguingly, TP53 splice mutations were associated with worse 5-year relapse-free survival in stage II disease, compared to both TP53 wild-type and exon mutations (P = 0.007). These data highlight the importance of including splice regions when examining the biological and clinical consequences of TP53 mutations in CRC

Inferior survival for patients with malignant peripheral nerve sheath tumors defined by aberrant TP53

Malignant peripheral nerve sheath tumor is a rare and aggressive disease with poor treatment response, mainly affecting adolescents and young adults. Few molecular biomarkers are used in the management of this cancer type, and although TP53 is one of few recurrently mutated genes in malignant peripheral nerve sheath tumor, the mutation prevalence and the corresponding clinical value of the TP53 network remains unsettled. We present a multi-level molecular study focused on aberrations in the TP53 network in relation to patient outcome in a series of malignant peripheral nerve sheath tumors from 100 patients and 38 neurofibromas, including TP53 sequencing, high-resolution copy number analyses of TP53 and MDM2, and gene expression profiling. Point mutations in TP53 were accompanied by loss of heterozygosity, resulting in complete loss of protein function in 8.2% of the malignant peripheral nerve sheath tumors. Another 5.5% had MDM2 amplification. TP53 mutation and MDM2 amplification were mutually exclusive and patients with either type of aberration in their tumor had a worse prognosis, compared to those without (hazard ratio for 5-year disease-specific survival 3.5, 95% confidence interval 1.78–6.98). Both aberrations had similar consequences on the gene expression level, as analyzed by a TP53-associated gene signature, a property also shared with the copy number aberrations and/or loss of heterozygosity at the TP53 locus, suggesting a common “TP53-mutated phenotype” in as many as 60% of the tumors. This was a poor prognostic phenotype (hazard ratio = 4.1, confidence interval:1.7–9.8), thus revealing a TP53-non-aberrant patient subgroup with a favorable outcome. The frequency of the “TP53-mutated phenotype” warrants explorative studies of stratified treatment strategies in malignant peripheral nerve sheath tumor