Transcriptome instability in colorectal cancer identified by exon microarray analyses: Associations with splicing factor expression levels and patient survival

Background Colorectal cancer (CRC) is a heterogeneous disease that, on the molecular level, can be characterized by inherent genomic instabilities; chromosome instability and microsatellite instability. In the present study we analyze genome-wide disruption of pre-mRNA splicing, and propose transcriptome instability as a characteristic that is analogous to genomic instability on the transcriptome level. Methods Exon microarray profiles from two independent series including a total of 160 CRCs were investigated for their relative amounts of exon usage differences. Each exon in each sample was assigned an alternative splicing score calculated by the FIRMA algorithm. Amounts of deviating exon usage per sample were derived from exons with extreme splicing scores. Results There was great heterogeneity within both series in terms of sample-wise amounts of deviating exon usage. This was strongly associated with the expression levels of approximately half of 280 splicing factors (54% and 48% of splicing factors were significantly correlated to deviating exon usage amounts in the two series). Samples with high or low amounts of deviating exon usage, associated with overall transcriptome instability, were almost completely separated into their respective groups by hierarchical clustering analysis of splicing factor expression levels in both sample series. Samples showing a preferential tendency towards deviating exon skipping or inclusion were associated with skewed transcriptome instability. There were significant associations between transcriptome instability and reduced patient survival in both sample series. In the test series, patients with skewed transcriptome instability showed the strongest prognostic association (P = 0.001), while a combination of the two characteristics showed the strongest association with poor survival in the validation series (P = 0.03). Conclusions We have described transcriptome instability as a characteristic of CRC. This transcriptome instability has associations with splicing factor expression levels and poor patient survival

Tumor Spreading to the Contralateral Ovary in Bilateral Ovarian Carcinoma Is a Late Event in Clonal Evolution

Cancer of the ovary is bilateral in 25%. Cytogenetic analysis could determine whether the disease in bilateral cases is metastatic or two separately occurring primary tumors, but karyotypic information comparing the two cancerous ovaries is limited to a single report with 11 informative cases. We present a series of 32 bilateral ovarian carcinoma cases, analyzed by karyotyping and high-resolution CGH. Our karyotypic findings showed that spreading to the contralateral ovary had occurred in bilateral ovarian cancer cases and that it was a late event in the clonal evolution of the tumors. This was confirmed by the large number of similar changes detected by HR-CGH in the different lesions from the same patient. The chromosomal bands most frequently involved in structural rearrangements were 19p13 (n = 12) and 19q13 (n = 11). The chromosomal bands most frequently gained by both tumorous ovaries were 5p14 (70%), 8q23-24 (65%), 1q23-24 (57%), and 12p12 (48%), whereas the most frequently lost bands were 17p11 (78%), 17p13 (74%), 17p12 (70%), 22q13 (61%), 8p21 and 19q13 (52%), and 8p22-23 (48%). This is the first time that 5p14 is seen gained at such a high frequency in cancer of the ovary; possibly oncogene(s) involved in bilateral ovarian carcinogenesis or tumor progression may reside in this band

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

Ovarian dysgerminomas are characterised by frequent KIT mutations and abundant expression of pluripotency markers

BACKGROUND: Ovarian germ cell tumours (OGCTs) typically arise in young females and their pathogenesis remains poorly understood. We investigated the origin of malignant OGCTs and underlying molecular events in the development of the various histological subtypes of this neoplasia. RESULTS: We examined in situ expression of stem cell-related (NANOG, OCT-3/4, KIT, AP-2γ) and germ cell-specific proteins (MAGE-A4, NY-ESO-1, TSPY) using a tissue microarray consisting of 60 OGCT tissue samples and eight ovarian small cell carcinoma samples. Developmental pattern of expression of NANOG, TSPY, NY-ESO-1 and MAGE-A4 was determined in foetal ovaries (gestational weeks 13–40). The molecular genetic part of our study included search for the presence of Y-chromosome material by fluorescence in situ hybridisation (FISH), and mutational analysis of the KIT oncogene (exon 17, codon 816), which is often mutated in testicular GCTs, in a subset of tumour DNA samples. We detected a high expression of transcription factors related to the embryonic stem cell-like pluripotency and undifferentiated state in OGCTs, but not in small cell carcinomas, supporting the view that the latter do not arise from a germ cell progenitor. Bilateral OGCTs expressed more stem cell markers than unilateral cases. However, KIT was mutated in 5/13 unilateral dysgerminomas, whereas all bilateral dysgerminomas (n = 4) and all other histological types (n = 22) showed a wild type sequence. Furthermore, tissue from five phenotypic female patients harbouring combined dysgerminoma/gonadoblastoma expressed TSPY and contained Y-chromosome material as confirmed by FISH. CONCLUSION: This study provides new data supporting two distinct but overlapping pathways in OGCT development; one involving spontaneous KIT mutation(s) leading to increased survival and proliferation of undifferentiated oogonia, the other related to presence of Y chromosome material and ensuing gonadal dysgenesis in phenotypic females

Optimality of broken extremals

In this paper we analyse the optimality of broken Pontryagin extremal for an n-dimensional affine control system with a control parameter, taking values in a k- dimensional closed ball. We prove the optimality of broken normal extremals when n = 3 and the controllable vector fields form a contact distribution, and when the Lie algebra of the controllable fields is locally orthogonal to the singular locus and the drift does not belong to it. Moreover, if k = 2, we show the optimality of any broken extremal even abnormal when the controllable fields do not form a contact distribution in the point of singularity.Comment: arXiv admin note: text overlap with arXiv:1610.0675

A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines

BACKGROUND: Tumor cell lines are commonly used as experimental tools in cancer research, but their relevance for the in vivo situation is debated. In a series of 11 microsatellite stable (MSS) and 9 microsatellite unstable (MSI) colon cancer cell lines and primary colon carcinomas (25 MSS and 28 MSI) with known ploidy stem line and APC, KRAS, and TP53 mutation status, we analyzed the promoter methylation of the following genes: hMLH1, MGMT, p16(INK4a )(CDKN2A α-transcript), p14(ARF )(CDKN2A β-transcript), APC, and E-cadherin (CDH1). We compared the DNA methylation profiles of the cell lines with those of the primary tumors. Finally, we examined if the epigenetic changes were associated with known genetic markers and/or clinicopathological variables. RESULTS: The cell lines and primary tumors generally showed similar overall distribution and frequencies of gene methylation. Among the cell lines, 15%, 50%, 75%, 65%, 20% and 15% showed promoter methylation for hMLH1, MGMT, p16(INK4a), p14(ARF), APC, and E-cadherin, respectively, whereas 21%, 40%, 32%, 38%, 32%, and 40% of the primary tumors were methylated for the same genes. hMLH1 and p14(ARF )were significantly more often methylated in MSI than in MSS primary tumors, whereas the remaining four genes showed similar methylation frequencies in the two groups. Methylation of p14(ARF), which indirectly inactivates TP53, was seen more frequently in tumors with normal TP53 than in mutated samples, but the difference was not statistically significant. Methylation of p14(ARF )and p16(INK4a )was often present in the same primary tumors, but association to diploidy, MSI, right-sided location and female gender was only significant for p14(ARF). E-cadherin was methylated in 14/34 tumors with altered APC further stimulating WNT signaling. CONCLUSIONS: The present study shows that colon cancer cell lines are in general relevant in vitro models, comparable with the in vivo situation, as the cell lines display many of the same molecular alterations as do the primary carcinomas. The combined pattern of epigenetic and genetic aberrations in the primary carcinomas reveals associations between them as well as to clinicopathological variables, and may aid in the future molecular assisted classification of clinically distinct stages

Digital image analysis of multiplex fluorescence IHC in colorectal cancer recognizes the prognostic value of CDX2 and its negative correlation with SOX2

Flourescence-based multiplex immunohistochemistry (mIHC) combined with multispectral imaging and digital image analysis (DIA) is a quantitative high-resolution method for determination of protein expression in tissue. We applied this method for five biomarkers (CDX2, SOX2, SOX9, E-cadherin, and beta-catenin) using tissue microarrays of a Norwegian unselected series of primary colorectal cancer. The data were compared with previously obtained chromogenic IHC data of the same tissue cores, visually assessed by the Allred method. We found comparable results between the methods, although confirmed that DIA offered improved resolution to differentiate cases with high and low protein expression. However, we experienced inherent challenges with digital image analysis of membrane staining, which was better assessed visually. DIA and mIHC enabled quantitative analysis of biomarker coexpression on the same tissue section at the single-cell level, revealing a strong negative correlation between the differentiation markers CDX2 and SOX2. Both methods confirmed known prognostic associations for CDX2, but DIA improved data visualization and detection of clinicopathological and biological associations. In summary, mIHC combined with DIA is an efficient and reliable method to evaluate protein expression in tissue, here shown to recapitulate and improve detection of known clinicopathological and survival associations for the emerging biomarker CDX2, and is therefore a candidate approach to standardize CDX2 detection in pathology laboratories.Peer reviewe

DNA methylation profiling of ovarian carcinomas and their in vitro models identifies HOXA9, HOXB5, SCGB3A1, and CRABP1 as novel targets

<p>Abstract</p> <p>Background</p> <p>The epigenetics of ovarian carcinogenesis remains poorly described. We have in the present study investigated the promoter methylation status of 13 genes in primary ovarian carcinomas (n = 52) and their <it>in vitro </it>models (n = 4; ES-2, OV-90, OVCAR-3, and SKOV-3) by methylation-specific polymerase chain reaction (MSP). Direct bisulphite sequencing analysis was used to confirm the methylation status of individual genes. The MSP results were compared with clinico- pathological features.</p> <p>Results</p> <p>Eight out of the 13 genes were hypermethylated among the ovarian carcinomas, and altogether 40 of 52 tumours were methylated in one or more genes. Promoter hypermethylation of <it>HOXA9</it>, <it>RASSF1A</it>, <it>APC</it>, <it>CDH13</it>, <it>HOXB5</it>, <it>SCGB3A1 (HIN-1)</it>, <it>CRABP1</it>, and <it>MLH1 </it>was found in 51% (26/51), 49% (23/47), 24% (12/51), 20% (10/51), 12% (6/52), 10% (5/52), 4% (2/48), and 2% (1/51) of the carcinomas, respectively, whereas <it>ADAMTS1</it>, <it>MGMT</it>, <it>NR3C1</it>, <it>p14</it>^<it>ARF</it>, and <it>p16</it>^{<it>INK</it>4<it>a </it>}were unmethylated in all samples. The methylation frequencies of <it>HOXA9 </it>and <it>SCGB3A1 </it>were higher among relatively early-stage carcinomas (FIGO I-II) than among carcinomas of later stages (FIGO III-IV; <it>P </it>= 0.002, <it>P </it>= 0.020, respectively). The majority of the early-stage carcinomas were of the endometrioid histotype. Additionally, <it>HOXA9 </it>hypermethylation was more common in tumours from patients older than 60 years of age (15/21) than among those of younger age (11/30; <it>P </it>= 0.023). Finally, there was a significant difference in <it>HOXA9 </it>methylation frequency among the histological types (<it>P </it>= 0.007).</p> <p>Conclusion</p> <p>DNA hypermethylation of tumour suppressor genes seems to play an important role in ovarian carcinogenesis and <it>HOXA9</it>, <it>HOXB5</it>, <it>SCGB3A1</it>, and <it>CRABP1 </it>are identified as novel hypermethylated target genes in this tumour type.</p

Distinct high resolution genome profiles of early onset and late onset colorectal cancer integrated with gene expression data identify candidate susceptibility loci

<p>Abstract</p> <p>Background</p> <p>Estimates suggest that up to 30% of colorectal cancers (CRC) may develop due to an increased genetic risk. The mean age at diagnosis for CRC is about 70 years. Time of disease onset 20 years younger than the mean age is assumed to be indicative of genetic susceptibility. We have compared high resolution tumor genome copy number variation (CNV) (Roche NimbleGen, 385 000 oligo CGH array) in microsatellite stable (MSS) tumors from two age groups, including 23 young at onset patients without known hereditary syndromes and with a median age of 44 years (range: 28-53) and 17 elderly patients with median age 79 years (range: 69-87). Our aim was to identify differences in the tumor genomes between these groups and pinpoint potential susceptibility loci. Integration analysis of CNV and genome wide mRNA expression data, available for the same tumors, was performed to identify a restricted candidate gene list.</p> <p>Results</p> <p>The total fraction of the genome with aberrant copy number, the overall genomic profile and the <it>TP53 </it>mutation spectrum were similar between the two age groups. However, both the number of chromosomal aberrations and the number of breakpoints differed significantly between the groups. Gains of 2q35, 10q21.3-22.1, 10q22.3 and 19q13.2-13.31 and losses from 1p31.3, 1q21.1, 2q21.2, 4p16.1-q28.3, 10p11.1 and 19p12, positions that in total contain more than 500 genes, were found significantly more often in the early onset group as compared to the late onset group. Integration analysis revealed a covariation of DNA copy number at these sites and mRNA expression for 107 of the genes. Seven of these genes, <it>CLC, EIF4E</it>, <it>LTBP4, PLA2G12A, PPAT</it>, <it>RG9MTD2</it>, and <it>ZNF574</it>, had significantly different mRNA expression comparing median expression levels across the transcriptome between the two groups.</p> <p>Conclusions</p> <p>Ten genomic loci, containing more than 500 protein coding genes, are identified as more often altered in tumors from early onset versus late onset CRC. Integration of genome and transcriptome data identifies seven novel candidate genes with the potential to identify an increased risk for CRC.</p