L1 hybridization enrichment: a method for directly accessing de novo L1 insertions in the human germline

Long interspersed nuclear element 1 (L1) retrotransposons are the only autonomously mobile human transposable elements. L1 retrotransposition has shaped our genome via insertional mutagenesis, sequence transduction, pseudogene formation, and ectopic recombination. However, L1 germline retrotransposition dynamics are poorly understood because de novo insertions occur very rarely: the frequency of disease‐causing retrotransposon insertions suggests that one insertion event occurs in roughly 18–180 gametes. The method described here recovers full‐length L1 insertions by using hybridization enrichment to capture L1 sequences from multiplex PCR‐amplified DNA. Enrichment is achieved by hybridizing L1‐specific biotinylated oligonucleotides to complementary molecules, followed by capture on streptavidin‐coated paramagnetic beads. We show that multiplex, long‐range PCR can amplify single molecules containing full‐length L1 insertions for recovery by hybridization enrichment. We screened 600 µg of sperm DNA from one donor, but no bone fide de novo L1 insertions were found, suggesting a L1 retrotransposition frequency of <1 insertion in 400 haploid genomes. This lies below the lower bound of previous estimates, and indicates that L1 insertion, at least into the loci studied, is very rare in the male germline. It is a paradox that L1 replication is ongoing in the face of such apparently low activity. Hum Mutat 32:1–11, 2011. © 2011 Wiley‐Liss, Inc.     

Reprogramming somatic cells into iPS cells activates LINE-1 retroelement mobility

Long interspersed element-1 (LINE-1 or L1) retrotransposons account for nearly 17% of human genomic DNA and represent a major evolutionary force that has reshaped the structure and function of the human genome. However, questions remain concerning both the frequency and the developmental timing of L1 retrotransposition in vivo and whether the mobility of these retroelements commonly results in insertional and post-insertional mechanisms of genomic injury. Cells exhibiting high rates of L1 retrotransposition might be especially at risk for such injury. We assessed L1 mRNA expression and L1 retrotransposition in two biologically relevant cell types, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), as well as in control parental human dermal fibroblasts (HDFs). Full-length L1 mRNA and the L1 open reading frame 1-encoded protein (ORF1p) were readily detected in hESCs and iPSCs, but not in HDFs. Sequencing analysis proved the expression of human-specific L1 element mRNAs in iPSCs. Bisulfite sequencing revealed that the increased L1 expression observed in iPSCs correlates with an overall decrease in CpG methylation in the L1 promoter region. Finally, retrotransposition of an engineered human L1 element was ~10-fold more efficient in iPSCs than in parental HDFs. These findings indicate that somatic cell reprogramming is associated with marked increases in L1 expression and perhaps increases in endogenous L1 retrotransposition, which could potentially impact the genomic integrity of the resultant iPSCs.     

Limited prognostic value of tissue protein expression levels of BCl‐2 in Danish ovarian cancer patients: from the Danish ‘MALOVA’ ovarian cancer study

Høgdall EVS, Christensen L, Kjaer SK, Blaakaer J, Christensen IJ, Høgdall CK. Limited prognostic value of tissue protein expression levels of BCl‐2 in Danish ovarian cancer patients. APMIS 2010; 118: 557–64. The purpose of the study was to determine the expression of BCl‐2 in epithelial ovarian tumors and to correlate expression levels with selected clinicopathologic parameters, time to progression and prognosis of the disease. Using tissue arrays (TA), we analyzed BCl‐2 expression in tissues from 191 women diagnosed with low malignant potential ovarian tumors (LMP) and from 582 patients diagnosed with ovarian cancer (OC). Using 30% as cutoff level for BCl‐2 overexpression, 5% of LMPs were positive with a higher proportion of serous ovarian tumor of LMP, compared to mucinous ovarian tumor of LMP (p = 0.02). Women with a BCl‐2‐positive LMP tumor were older than women with a BCl‐2 negative tumor (p = 0.02). Ten percent of OCs were positive for BCl‐2 expression (≥30%). No significant association was found between BCl‐2 expression levels and histologic type of tumors (serous vs mucinous, p = 0.19). A 30% cutoff value or a percentage scale showed that BCl‐2 expression had no prognostic value, both in univariate and in multivariate survival analyses. No difference in time to progression was observed between patients with BCl‐2‐positive and negative tumors. These data suggest that BCl‐2 expression may not be of important clinical value in the treatment of Danish OC patients.     

Overexpression and gene amplification of both ERBB2 and EGFR in an esophageal squamous cell carcinoma revealed by fluorescence in situ hybridization,multiplex ligation‐dependent probe amplification and immunohistochemistry

EGFR and ERBB2 belong to the EGFR gene family. In esophageal squamous cell carcinomas (SCCs), amplification of EGFR or ERBB2 is usually mutually exclusive. EGFR amplification occurs in approximately 15% of SCCs, ERBB2 occurs in less than 5%. Here, we report the co‐amplification of EGFR and ERBB2 in an ulcerative and infiltrating‐type SCC that measured approximately 4.2 × 2.7 × 1.2 cm with a superficial lesion occurring in the thoracic esophagus of a 72‐year‐old man. Multiplex ligation‐dependent probe amplification using representative tumor sections showed gain of CCND1 and coincident amplification of ERBB2 or EGFR or neither. Immunohistochemistry and fluorescence in situ hybridization revealed that the tumor comprised three cancer‐cell populations: well‐differentiated SCC with high‐level ERBB2 amplification and ERBB2 overexpression, more infiltrative poorly‐differentiated SCC with high‐level EGFR amplification and EGFR overexpression, and poorly‐differentiated SCC lacking any ERBB2 or EGFR abnormality. These three populations each had low‐level CCND1 amplification and nuclear cyclin D1 overexpression. This histological topology and gene amplification combinations suggested that genetic instability first produced CCND1 amplification, and then ERBB2 or EGFR gene amplification occurred. It is further speculated that during cancer progression and clonal selection indecisive predominance of either clone caused the rare co‐amplification of ERBB2 and EGFR in a single chimeric tumor.     

L1 retrotransposition occurs mainly in embryogenesis and creates somatic mosaicism

Long Interspersed Element 1 (L1) is a retrotransposon that comprises ∼17% of the human genome. Despite its abundance in mammalian genomes, relatively little is understood about L1 retrotransposition in vivo. To study the timing and tissue specificity of retrotransposition, we created transgenic mouse and rat models containing human or mouse L1 elements controlled by their endogenous promoters. Here, we demonstrate abundant L1 RNA in both germ cells and embryos. However, the integration events usually occur in embryogenesis rather than in germ cells and are not heritable. We further demonstrate L1 RNA in preimplantation embryos lacking the L1 transgene and L1 somatic retrotransposition events in blastocysts and adults lacking the transgene. Together, these data indicate that L1 RNA transcribed in male or female germ cells can be carried over through fertilization and integrate during embryogenesis, an interesting example of heritability of RNA independent of its encoding DNA. Thus, L1 creates somatic mosaicism during mammalian development, suggesting a role for L1 in carcinogenesis and other disease.     

Widespread somatic L1 retrotransposition occurs early during gastrointestinal cancer evolution

Somatic L1 retrotransposition events have been shown to occur in epithelial cancers. Here, we attempted to determine how early somatic L1 insertions occurred during the development of gastrointestinal (GI) cancers. Using L1-targeted resequencing (L1-seq), we studied different stages of four colorectal cancers arising from colonic polyps, seven pancreatic carcinomas, as well as seven gastric cancers. Surprisingly, we found somatic L1 insertions not only in all cancer types and metastases but also in colonic adenomas, well-known cancer precursors. Some insertions were also present in low quantities in normal GI tissues, occasionally caught in the act of being clonally fixed in the adjacent tumors. Insertions in adenomas and cancers numbered in the hundreds, and many were present in multiple tumor sections, implying clonal distribution. Our results demonstrate that extensive somatic insertional mutagenesis occurs very early during the development of GI tumors, probably before dysplastic growth.Somatic mobilization of retroelements in the cancer genome has only recently been established as a widespread mutational phenomenon. In particular, Long INterspersed Element (LINE)-1 (L1)-mediated retrotransposition has been observed mostly in epithelial cancers. Somatic human-specific L1 (L1Hs) insertions are most abundant in these cancers, but L1-mediated Alu, SVA, and processed pseudogene insertions have also been detected (Iskow et al. 2010; Lee et al. 2012; Solyom et al. 2012; Ewing et al. 2013; Shukla et al. 2013; Cooke et al. 2014; Helman et al. 2014; Pitkanen et al. 2014; Tubio et al. 2014). L1s are autonomous mobile elements that comprise 17% of the human genome and retrotranspose by a “copy and paste” mechanism via an RNA intermediate. This process can lead to insertional mutagenesis and genetic instability (Goodier and Kazazian 2008). Potentially etiological L1 insertions have been reported in APC (Miki et al. 1992) and PTEN exons (Helman et al. 2014) in colorectal and endometrial cancer, respectively, and insertions of unknown significance have been found in numerous other cancer driver genes in a variety of malignancies (Iskow et al. 2010; Lee et al. 2012; Solyom et al. 2012; Ewing et al. 2013; Shukla et al. 2013; Cooke et al. 2014; Helman et al. 2014; Pitkanen et al. 2014; Tubio et al. 2014; Paterson et al. 2015). In a study of somatic retrotransposition during the evolution of prostate and lung cancer, Tubio et al. (2014) found evidence of insertions occurring during cancer development. Beyond this work, the timing of retrotransposition in cancer development has not been analyzed previously.     

Prognostic significance of neuroendocrine differentiation, proliferation activity and androgen receptor expression in prostate cancer

Androgen, acting via the androgen receptor (AR), is associated with the development and progression of prostate cancer. Anti-androgen therapy is widely used to manage prostate cancer. However, the conversion of the tumor from a hormone-sensitive to a hormone-insensitive status causes such therapy to fail. Several mechanisms have now been put forward for this conversion, including neuroendocrine (NE) differentiation of the tumor cells. In this study, we evaluated the prognostic significance of tumor-cell proliferation activity, NE differentiation and AR expression. Formalin-fixed, paraffin-embedded sections were prepared from 42 patients with adenocarcinoma of the prostate. Using antibodies to AR, the Ki-67 antigen (MIB-1), chromogranin A and synaptophysin, immunohistochemical expression of AR, tumor proliferation activity and NE differentiation were analyzed. Our study revealed that AR expression was significantly lower in adenocarcinoma (52.2 +/- 27.1%) than in non-tumorous prostate tissue (68.3 +/- 18.3%; P < 0.001). NE differentiation was found in 50% of the tumors, which was correlated with the Gleason score (P < 0.05). An univariate analysis revealed a significant correlation between progression-free survival with both AR expression (P < 0.01) and proliferation activity (P < 0.001). NE differentiation was not a prognostic factor in this study.     

Somatic Alterations Contributing to Metastasis of a Castration‐Resistant Prostate Cancer

Metastatic castration‐resistant prostate cancer (mCRPC) is a lethal disease, and molecular markers that differentiate indolent from aggressive subtypes are needed. We sequenced the exomes of five metastatic tumors and healthy kidney tissue from an index case with mCRPC to identify lesions associated with disease progression and metastasis. An Ashkenazi Jewish (AJ) germline founder mutation, del185AG in BRCA1, was observed and AJ ancestry was confirmed. Sixty‐two somatic variants altered proteins in tumors, including cancer‐associated genes, TMPRSS2‐ERG, PBRM1, and TET2. The majority (n = 53) of somatic variants were present in all metastases and only a subset (n = 31) was observed in the primary tumor. Integrating tumor next‐generation sequencing and DNA copy number showed somatic loss of BRCA1 and TMPRSS2‐ERG. We sequenced 19 genes with deleterious mutations in the index case in additional mCRPC samples and detected a frameshift, two somatic missense alterations, tumor loss of heterozygosity, and combinations of germline missense SNPs in TET2. In summary, genetic analysis of metastases from an index case permitted us to infer a chronology for the clonal spread of disease based on sequential accrual of somatic lesions. The role of TET2 in mCRPC deserves additional analysis and may define a subset of metastatic disease.     

Genetic diagnosis of familial breast cancer using clonal sequencing

Using conventional Sanger sequencing as a reference standard, we compared the sensitivity, specificity, and capacity of the Illumina GA II platform for the detection of TP53, BRCA1, and BRCA2 mutations in established tumor cell lines and DNA from patients with germline mutations. A total of 656 coding variants were identified in four cell lines and 65 patient DNAs. All of the known pathogenic mutations (including point mutations and insertions/deletions of up to 16 nucleotides) were identified, using a combination of the Illumina data analysis pipeline with custom and commercial sequence alignment software. In our configuration, clonal sequencing outperforms current diagnostic methods, providing a reduction in analysis times and in reagent costs compared with conventional sequencing. These improvements open the possibility of BRCA1/2 testing for a wider spectrum of at‐risk women, and will allow the genetic classification of tumors prior to the use of novel PARP inhibitors to treat BRCA‐deficient breast cancers. Hum Mutat 31:1–8, 2010. © 2010 Wiley‐Liss, Inc.     

Unconventional translation of mammalian LINE-1 retrotransposons

Long Interspersed Element-1 (LINE-1 or L1) retrotransposons encode proteins required for their mobility (ORF1p and ORF2p), yet little is known about how L1 mRNA is translated. Here, we show that ORF2 translation generally initiates from the first in-frame methionine codon of ORF2, and that both ORF1 and the inter-ORF spacer are dispensable for ORF2 translation. Remarkably, changing the ORF2 AUG codon to any other coding triplet is compatible with retrotransposition. However, introducing a premature termination codon in ORF1 or a thermostable hairpin in the inter-ORF spacer reduces ORF2p translation or L1 retrotransposition to approximately 5% of wild-type levels. Similar data obtained from "natural" and codon optimized "synthetic" mouse L1s lead us to propose that ORF2 is translated by an unconventional termination/reinitiation mechanism.