Testicular germ cell tumours (GCTs) of adolescents and adults can be subdivided into seminomas (referred to as dysgerminomas of the ovary) and non-seminomas, all referred to as type II GCTs. They originate from carcinoma in situ (CIS), being the malignant counterparts of primordial germ cells (PGCs)/gonocytes. The invasive components mimic embryogenesis, including the stem cell component embryonal carcinoma (EC), the somatic lineage teratoma (TE), and the extra-embryonic tissues yolk sac tumour (YST) and choriocarcinoma (CH). The other type is the so-called spermatocytic seminomas (SS, type III GCT), composed of neoplastic primary spermatocytes. We reported previously that the miRNAs hsa-miR 371-373 cluster is involved in overruling cellular senescence induced by oncogenic stress, allowing cells to become malignant. Here we report the first high-throughput screen of 156 microRNAs in a series of type II and III GCTs (n = 69, in duplicate) using a quantitative PCR-based approach. After normalization to allow inter-sample analysis, the technical replicates clustered together, and the previous hsa-miRNA 371-373 cluster finding was confirmed. Unsupervised cluster analysis demonstrated that the cell lines are different from the in vivo samples. The in vivo samples, both normal and malignant, clustered predominantly based on their maturation status. This parallels normal embryogenesis, rather than chromosomal anomalies in the tumours. miRNAs within a single cluster showed a similar expression pattern, implying common regulatory mechanisms. Normal testicular tissue expressed most discriminating miRNAs at a higher level than SE and SS. Moreover, differentiated non-seminomas showed overexpression of discriminating miRNAs. These results support the model that miRNAs are involved in regulating differentiation of stem cells, retained in GCTs.

Testicular germ cell tumors of adolescents and adults (TGCTs) can be classified into seminomatous and nonseminomatous tumors. Various nonseminomatous cell lines, predominantly embryonal carcinoma, have been established and proven to be valuable for pathobiological and clinical studies. So far, no cell lines have been derived from seminoma which constitutes more than 50% of invasive TGCTs. Such a cell line is essential for experimental investigation of biological characteristics of the cell of origin of TGCTs, i.e., carcinoma in situ of the testis, which shows characteristics of a seminoma cell. Before a cell line can be used as model, it must be verified regarding its origin and characteristics. Therefore, a multidisciplinary approach was undertaken on TCam-2 cells, supposedly the first seminoma cell line. Fluorescence in situ hybridization, array comparative genomic hybridization, and spectral karyotyping demonstrated an aneuploid DNA content, with gain of 12p, characteristic for TGCTs. Genome wide mRNA and microRNA expression profiling supported the seminoma origin, in line with the biallelic expression of imprinted genes IGF2/H19 and associated demethylation of the imprinting control region. Moreover, the presence of specific markers, demonstrated by immunohistochemistry, including (wild type) KIT, stem cell factor, placental alkaline phosphatase, OCT3/4 (also demonstrated by a specific Q-PCR) and NANOG, and the absence of CD30, SSX2-4, and SOX2, confirms that TCam-2 is a seminoma cell line. Although mutations in oncogenes and tumor suppressor genes are rather rare in TGCTs, TCam-2 had a mutated BRAF gene (V600E), which likely explains the fact that these cells could be propagated in vitro. In conclusion, TCam-2 is the first well-characterized seminoma-derived cell line, with an exceptional mutation, rarely found in TGCTs.

Molecular genetic analysis of isolated single cells and other minute DNA samples is limited because there is insufficient DNA to perform more than one independent PCR amplification. One solution to this problem is to first amplify the entire genome, thus providing enough DNA for numerous subsequent PCRs. In this study we have investigated four different methods of whole genome amplification performed on single cells, and have identified a protocol that generates sufficient quantities of DNA for comparative genomic hybridisation (CGH) as well as more than 90 independent amplification reactions. Thus, numerous specific loci and the copy number of every chromosome can be assessed in a single cell. We report here the first reliable application of CGH to single cells from human preimplantation embryos (blastomeres) and to single fibroblasts, buccal cells and amniocytes.

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NGS evaluation with the Oncomine Solid Tumour Fusion transcript kit and RT-PCR proved to have high sensitivity and specificity, advocating their use in routine practice. For maximal sensitivity and specificity, ALK status should be assessed by using two techniques and a third one in discordant cases. We therefore propose a customizable testing algorithm. These findings significantly influence existing testing paradigms and have clear clinical and economic impact.

SummaryBackgroundMolecular indicators of colorectal cancer prognosis have been assessed in several studies, but most analyses have been restricted to a handful of markers. We aimed to identify prognostic biomarkers for colorectal cancer by sequencing panels of multiple driver genes.MethodsIn stage II or III colorectal cancers from the QUASAR 2 open-label randomised phase 3 clinical trial and an Australian community-based series, we used targeted next-generation sequencing of 82 and 113 genes, respectively, including the main colorectal cancer drivers. We investigated molecular pathways of tumorigenesis, and analysed individual driver gene mutations, combinations of mutations, or global measures such as microsatellite instability (MSI) and mutation burden (total number of non-synonymous mutations and coding indels) for associations with relapse-free survival in univariable and multivariable models, principally Cox proportional hazards models.FindingsIn QUASAR 2 (511 tumours), TP53, KRAS, BRAF, and GNAS mutations were independently associated with shorter relapse-free survival (p<0·035 in all cases), and total somatic mutation burden with longer survival (hazard ratio [HR] 0·81 [95% CI 0·68–0·96]; p=0·014). MSI was not independently associated with survival (HR 1·12 [95% CI 0·57–2·19]; p=0·75). We successfully validated these associations in the Australian sample set (296 tumours). In a combined analysis of both the QUASAR 2 and the Australian sample sets, mutation burden was also associated with longer survival (HR 0·84 [95% CI 0·74–0·94]; p=0·004) after exclusion of MSI-positive and POLE mutant tumours. In an extended analysis of 1732 QUASAR 2 and Australian colorectal cancers for which KRAS, BRAF, and MSI status were available, KRAS and BRAF mutations were specifically associated with poor prognosis in MSI-negative cancers. MSI-positive cancers with KRAS or BRAF mutations had better prognosis than MSI-negative cancers that were wild-type for KRAS or BRAF. Mutations in the genes NF1 and NRAS from the MAPK pathway co-occurred, and mutations in the DNA damage-response genes TP53 and ATM were mutually exclusive. We compared a prognostic model based on the gold standard of clinicopathological variables and MSI with our new model incorporating clinicopathological variables, mutation burden, and driver mutations in KRAS, BRAF, and TP53. In both QUASAR 2 and the Australian cohort, our new model was significantly better (p=0·00004 and p=0·0057, respectively, based on a likelihood ratio test).InterpretationMultigene panels identified two previously unreported prognostic associations in colorectal cancer involving TP53 mutation and total mutation burden, and confirmed associations with KRAS and BRAF. Even a modest-sized gene panel can provide important information for use in clinical practice and outperform MSI-based prognostic models.FundingUK Technology Strategy Board, National Institute for Health Research Oxford Biomedical Research Centre, Cancer Australia Project, Cancer Council Victoria, Ludwig Institute for Cancer R...

Aneuploidies involving chromosomes 21, 18, 13, X and Y account for over 95% of all chromosomal abnormalities in live-born infants. Prenatal diagnosis of these disorders is usually accomplished by cytogenetic analysis of amniotic or chorionic cells but this is a lengthy procedure requiring great technical expertise. In this paper, we assess the diagnostic value of using a quantitative fluorescent polymerase chain reaction (PCR) suitable for the simultaneous and rapid diagnosis of trisomies 21 and 18 together with the detection of DNA sequences derived from the X and Y chromosomes. Samples of DNA, extracted from amniotic fluid, fetal blood or tissues, and peripheral blood from normal adults were investigated by quantitative fluorescent PCR amplification of polymorphic small tandem repeats (STRs) specific for two loci on each of chromosomes 21 and 18. Quantitative analysis of the amplification products allowed the diagnosis of trisomies 21 and 18, while sexing was performed simultaneously using PCR amplification of DNA sequences derived from the chromosomes X and Y. These results indicate the advantages of using two sets of STR markers for the detection of chromosome 21 trisomies and confirmed the usefulness of quantitative fluorescent multiplex PCR for the rapid prenatal diagnosis of selected chromosomal abnormalities.

Quantitative fluorescent polymerase chain reaction (QF‐PCR) assays and small tandem repeat (STR) markers have been successfully employed for the rapid detection of major numerical aneuploidies affecting human autosomes. So far, the analysis of chromosomes X and Y disorders has been hampered by the rarity of highly polymorphic markers which could distinguish normal female homozygous PCR patterns from those seen in patients with Turner's syndrome. A new marker (X22) of the X/Y chromosomes has been identified which maps in the Xq/Yq pseudoautosomal region PAR2; used together with the HPRT it allows the rapid diagnosis of numerical aneuploidies of the sex chromosomes. Blood samples from normal male and female subjects and from patients with X and Y chromosome disorders (45,X and 47,XXY) have been tested by QF‐PCR with the X22 polymorphic pentanucleotide (12 alleles) together with the HPRT and P39 markers. The samples were also tested by multiplex QF‐PCR with STRs specific for chromosomes 21,18,13 and amelogenin (AMXY). Tested by QF‐PCR, all samples from normal females were heterozygous for either the X22 or the HPRT marker with fluorescent peak ratios near 1:1, thus allowing a correct, rapid diagnosis of their chromosome complement. Turner's patients (45,X) showed only one X22 and one HPRT fluorescent peak, thus documenting the presence of a single X chromosome. Turner's patients with mosaicism showed a major fluorescent peak for the X22 and HPRT markers and a minor peak revealing the presence of a second minor population of cells. Two 47,XXY cases could also be diagnosed. Multiplex analyses can be performed using simultaneously STR markers for chromosomes 21,18,13 X and Y. The diagnostic value of a third X‐linked marker (P39) was also investigated. These results suggest that rapid diagnosis of major numerical anomalies of the X and Y chromosomes can be performed using QF‐PCR with a new highly polymorphic X‐linked marker, X22, which maps in the Xq/Yq pseudoautosomal region PAR 2. Multiplex QF‐PCR tests—using the X22 STR in association with HPRT and, in rare cases, a third P39 marker—allow the rapid diagnosis of major aneuploidies affecting chromosomes 21, 18, 13, X and Y. The X22 marker can also be employed for the detection of fetal cells present in maternal peripheral blood or the endocervical canal. Copyright © 1999 John Wiley & Sons, Ltd.