Intra-patient Inter-metastatic Genetic Heterogeneity in Colorectal Cancer as a Key Determinant of Survival after Curative Liver Resection.

Chromosomal instability is a well-defined hallmark of tumor aggressiveness and metastatic progression in colorectal cancer. The magnitude of genetic heterogeneity among distinct liver metastases from the same patient at the copy number level, as well as its relationship with chemotherapy exposure and patient outcome, remains unknown. We performed high-resolution DNA copy number analyses of 134 liver metastatic deposits from 45 colorectal cancer patients to assess: (i) intra-patient inter-metastatic genetic heterogeneity using a heterogeneity score based on pair-wise genetic distances among tumor deposits; and (ii) genomic complexity, defined as the proportion of the genome harboring aberrant DNA copy numbers. Results were analyzed in relation to the patients' clinical course; previous chemotherapy exposure and outcome after surgical resection of liver metastases. We observed substantial variation in the level of intra-patient inter-metastatic heterogeneity. Heterogeneity was not associated with the number of metastatic lesions or their genomic complexity. In metachronous disease, heterogeneity was higher in patients previously exposed to chemotherapy. Importantly, intra-patient inter-metastatic heterogeneity was a strong prognostic determinant, stronger than known clinicopathological prognostic parameters. Patients with a low level of heterogeneity (below the median level) had a three-year progression-free and overall survival rate of 23% and 66% respectively, versus 5% and 18% for patients with a high level (hazard ratio0.4, 95% confidence interval 0.2-0.8, P = 0.01; and hazard ratio0.3,95% confidence interval 0.1-0.7, P = 0.007). A low patient-wise level of genomic complexity (below 25%) was also a favorable prognostic factor; however, the prognostic association of intra-patient heterogeneity was independent of genomic complexity in multivariable analyses. In conclusion, intra-patient inter-metastatic genetic heterogeneity is a pronounced feature of metastatic colorectal cancer, and the strong prognostic association reinforces its clinical relevance and places it as a key feature to be explored in future patient cohorts.

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.

Transcriptomic subtyping of malignant peripheral nerve sheath tumours highlights immune signatures, genomic profiles, patient survival and therapeutic targets.

BACKGROUND: Malignant peripheral nerve sheath tumour (MPNST) is an aggressive orphan disease commonly affecting adolescents or young adults. Current knowledge of molecular tumour biology has been insufficient for development of rational treatment strategies. We aimed to discover molecular subtypes of potential clinical relevance. METHODS: Fresh frozen samples of MPNSTs (n = 94) and benign neurofibromas (n = 28) from 115 patients in a European multicentre study were analysed by DNA copy number and/or transcriptomic profiling. Unsupervised transcriptomic subtyping was performed and the subtypes characterized for genomic aberrations, clinicopathological associations and patient survival. FINDINGS: MPNSTs were classified into two transcriptomic subtypes defined primarily by immune signatures and proliferative processes. "Immune active" MPNSTs (44%) had sustained immune signals relative to neurofibromas, were more frequently low-grade (P = 0.01) and had favourable prognostic associations in a multivariable model of disease-specific survival with clinicopathological factors (hazard ratio 0.25, P = 0.003). "Immune deficient" MPNSTs were more aggressive and characterized by proliferative signatures, high genomic complexity, aberrant TP53 and PRC2 loss, as well as high relative expression of several potential actionable targets (EGFR, ERBB2, EZH2, KIF11, PLK1, RRM2). Integrated gene-wise analyses suggested a DNA copy number-basis for proliferative transcriptomic signatures in particular, and the tumour copy number burden further stratified the transcriptomic subtypes according to patient prognosis (P < 0.01). INTERPRETATION: Approximately half of MPNSTs belong to an "immune deficient" transcriptomic subtype associated with an aggressive disease course, PRC2 loss and expression of several potential therapeutic targets, providing a rationale for molecularly-guided intervention trials. FUNDING: Research grants from non-profit organizations, as stated in the Acknowledgements.

Frequent copy number gains of SLC2A3 and ETV1 in testicular embryonal carcinomas.

Testicular germ cell tumours (TGCTs) appear as different histological subtypes or mixtures of these. They show similar, multiple DNA copy number changes, where gain of 12p is pathognomonic. However, few high-resolution analyses have been performed and focal DNA copy number changes with corresponding candidate target genes remain poorly described for individual subtypes. We present the first high-resolution DNA copy number aberration (CNA) analysis on the subtype embryonal carcinomas (ECs), including 13 primary ECs and 5 EC cell lines. We identified recurrent gains and losses and allele-specific CNAs. Within these regions, we nominate 30 genes that may be of interest to the EC subtype. By in silico analysis of data from 150 TGCTs from The Cancer Genome Atlas (TCGA), we further investigated CNAs, RNA expression, somatic mutations and fusion transcripts of these genes. Among primary ECs, ploidy ranged between 2.3 and 5.0, and the most common aberrations were DNA copy number gains at chromosome (arm) 7, 8, 12p, and 17, losses at 4, 10, 11, and 18, replicating known TGCT genome characteristics. Gain of whole or parts of 12p was found in all samples, including a highly amplified 100 kbp segment at 12p13.31, containing SLC2A3. Gain at 7p21, encompassing ETV1, was the second most frequent aberration. In conclusion, we present novel CNAs and the genes located within these regions, where the copy number gain of SLC2A3 and ETV1 are of interest, and which copy number levels also correlate with expression in TGCTs.

Gene expression profiles of CMS2-epithelial/canonical colorectal cancers are largely driven by DNA copy number gains.

About 80% of colorectal cancers (CRCs) have chromosomal instability, which is an integral part of aggressive malignancy development, but the importance of specific copy number aberrations (CNAs) in modulating gene expression, particularly within the framework of clinically relevant molecular subtypes, remains mostly elusive. We performed DNA copy number profiling of 257 stage I-IV primary CRCs and integrative gene expression analysis in 151 microsatellite stable (MSS) tumors, focusing on high-level amplifications and the effect of CNAs on the characteristics of the gene expression-based consensus molecular subtypes (CMS). The results were validated in 323 MSS tumors from TCGA. Novel recurrent high-level amplifications (≥15 additional copies) with a major impact on gene expression were found for TOX3 (16q) at 1.5% frequency, as well as for CCND2 (12p) and ANXA11 (10q) at 1% frequency, in addition to the well-known targets ERBB2 (17q) and MYC (8q). Focal amplifications with ≥15 or ≥5 additional copies of at least one of these regions were associated with a poor overall survival among patients with stage I-III MSS CRCs (multivariable hazard ratio ≥3.2, p ≤ 0.01). All high-level amplifications were focal and had a more consistent relationship with gene expression than lower amplitude and/or broad-range amplifications, suggesting specific targeting during carcinogenesis. Genome-wide, copy number driven gene expression was enriched for pathways characteristic of the CMS2-epithelial/canonical subtype, including DNA repair and cell cycle progression. Furthermore, 50% of upregulated genes in CMS2-epithelial/canonical MSS CRCs were driven by CNAs, an enrichment compared with the other CMS groups, and associated with the stronger correspondence between CNAs and gene expression in malignant epithelial cells than in the cells of the tumor microenvironment (fibroblasts, endothelial cells, leukocytes). In conclusion, we identify novel recurrent amplifications with impact on gene expression in CRC and provide the first evidence that CMS2 may have a stronger copy-number related genetic basis than subtypes more heavily influenced by gene expression signals from the tumor microenvironment.

Drug sensitivity and resistance testing identifies PLK1 inhibitors and gemcitabine as potent drugs for malignant peripheral nerve sheath tumors.

Patients with malignant peripheral nerve sheath tumor (MPNST), a rare soft tissue cancer associated with loss of the tumor suppressor neurofibromin (NF1), have poor prognosis and typically respond poorly to adjuvant therapy. We evaluated the effect of 299 clinical and investigational compounds on seven MPNST cell lines, two primary cultures of human Schwann cells, and five normal bone marrow aspirates, to identify potent drugs for MPNST treatment with few side effects. Top hits included Polo-like kinase 1 (PLK1) inhibitors (volasertib and BI2536) and the fluoronucleoside gemcitabine, which were validated in orthogonal assays measuring viability, cytotoxicity, and apoptosis. DNA copy number, gene expression, and protein expression were determined for the cell lines to assess pharmacogenomic relationships. MPNST cells were more sensitive to BI2536 and gemcitabine compared to a reference set of 94 cancer cell lines. PLK1, RRM1, and RRM2 mRNA levels were increased in MPNST compared to benign neurofibroma tissue, and the protein level of PLK1 was increased in the MPNST cell lines compared to normal Schwann cells, indicating an increased dependence on these drug targets in malignant cells. Furthermore, we observed an association between increased mRNA expression of PLK1, RRM1, and RRM2 in patient samples and worse disease outcome, suggesting a selective benefit from inhibition of these genes in the most aggressive tumors.

Protein expression of BIRC5, TK1, and TOP2A in malignant peripheral nerve sheath tumours--A prognostic test after surgical resection.

No consensus treatment regime exists beyond surgery for malignant peripheral nerve sheath tumours (MPNST), and the purpose of the present study was to find new approaches to stratify patients with good and poor prognosis and to better guide therapeutic intervention for this aggressive soft tissue cancer. From a total of 67 MPNSTs from Scandinavian patients with and without neurofibromatosis type 1, 30 MPNSTs were investigated by genome-wide RNA expression profiling and 63 MPNSTs by immunohistochemical (IHC) analysis, and selected genes were submitted to analyses of disease-specific survival. The potential drug target genes survivin (BIRC5), thymidine kinase 1 (TK1), and topoisomerase 2-alpha (TOP2A), all encoded on chromosome arm 17q, were up-regulated in MPNST as compared to benign neurofibromas. Each of them was found to be independent prognostic markers on the gene expression level, as well as on the protein level. A prognostic profile was identified by combining the nuclear expression scores of the three proteins. For patients with completely resected tumours only 15% in the high risk group were alive after two years, as compared to 78% in the low risk group. In conclusion, we found a novel protein expression profile which identifies MPNST patients with inferior prognosis even after assumed curative surgery. The tested proteins are drug targets; therefore the expression profile may provide predictive information guiding the design of future clinical trials. Importantly, as the effect is seen on the protein level using IHC, the biomarker panel can be readily implemented in routine clinical testing.

Methylated RASSF1A in malignant peripheral nerve sheath tumors identifies neurofibromatosis type 1 patients with inferior prognosis.

BACKGROUND: Malignant peripheral nerve sheath tumor (MPNST) is a rare and highly aggressive disease with no evidence of effect from adjuvant therapy. It is further associated with the hereditary syndrome neurofibromatosis type 1 (NF1). Silencing of the tumor suppressor gene RASSF1A through DNA promoter hypermethylation is known to be involved in cancer development, but its impact in MPNSTs remains unsettled. METHODS: The RASSF1A promoter was analyzed by methylation-specific PCR in 113 specimens, including 44 NF1-associated MPNSTs, 47 sporadic MPNSTs, 21 benign neurofibromas, and 1 nonneoplastic nerve sheath control. RESULTS: RASSF1A methylation was found only in the malignant samples (60%) and identified a subgroup among patients with NF1-associated MPNST with a poor prognosis. These patients had a mean 5-year disease-specific survival of 27.3 months (95% CI: 17.2-37.4) versus 47.4 months (95% CI: 37.5-57.2) for NF1 patients with unmethylated promoters, P = 0.014. In multivariate Cox regression analysis, methylated RASSF1A remained an adverse prognostic factor independent of clinical risk factors, P = .013 (hazard ratio: 5.2; 95% CI: 1.4-19.4). CONCLUSION: A considerable number of MPNST samples display hypermethylation of the RASSF1A gene promoter, and for these tumors, this is the first molecular marker that if validated can characterize a subgroup of patients with inferior prognosis, restricted to individuals with NF1.

Survival meta-analyses for >1800 malignant peripheral nerve sheath tumor patients with and without neurofibromatosis type 1.

There are conflicting reports as to whether malignant peripheral nerve sheath tumor (MPNST) patients with neurofibromatosis type 1 (NF1) have worse prognosis than non-NF1 MPNST patients. Large clinical studies to address this problem are lacking due to the rareness of MPNST. We have performed meta-analyses testing the effect of NF1 status on MPNST survival based on publications from the last 50 years, including only nonoverlapping patients reported from each institution. In addition, we analyzed survival characteristics for 179 MPNST patients from 3 European sarcoma centers. The meta-analyses including data from a total of 48 studies and >1800 patients revealed a significantly higher odds ratio for overall survival (OR(OS)) and disease-specific survival (OR(DSS)) in the non-NF1 group (OR(OS) = 1.75, 95% confidence interval [CI] = 1.28-2.39, and OR(DSS) = 1.68, 95% CI = 1.18-2.40). However, in studies published in the last decade, survival in the 2 patient groups has been converging, as especially the NF1 group has shown improved prognosis. For our own MPNST patients, NF1 status had no effect on overall or disease-specific survival. The compiled literature from 1963 to the present indicates a significantly worse outcome of MPNST in patients with NF1 syndrome compared with non-NF1 patients. However, survival for the NF1 patients has improved in the last decade, and the survival difference is diminishing. These observations support the hypothesis that MPNSTs arising in NF1 and non-NF1 patients are not different per se. Consequently, we suggest that the choice of treatment for MPNST should be independent of NF1 status.