A new workflow for whole-genome sequencing of single human cells.

Unbiased amplification of the whole-genome amplification (WGA) of single cells is crucial to study cancer evolution and genetic heterogeneity, but is challenging due to the high complexity of the human genome. Here, we present a new workflow combining an efficient adapter-linker PCR-based WGA method with second-generation sequencing. This approach allows comparison of single cells at base pair resolution. Amplification recovered up to 74% of the human genome. Copy-number variants and loss of heterozygosity detected in single cell genomes showed concordance of up to 99% to pooled genomic DNA. Allele frequencies of mutations could be determined accurately due to an allele dropout rate of only 2%, clearly demonstrating the low bias of our PCR-based WGA approach. Sequencing with paired-end reads allowed genome-wide analysis of structural variants. By direct comparison to other WGA methods, we further endorse its suitability to analyze genetic heterogeneity.

EPCAM-A novel molecular target for the treatment of pediatric and adult germ cell tumors.

Germ cell tumors (GCTs) are thought to develop from totipotent primordial germ cells. Although the epithelial cell adhesion molecule (EPCAM) is expressed on embryonic stem cells as well as different tumor cells, it has not yet been extensively studied in GCTs. We analyzed EPCAM expression by quantitative RT-PCR in 48 fresh-frozen GCT specimens of different histology (10 mature teratoma, MT; 6 immature teratoma, IT; 7 dysgerminoma; 6 mixed malignant GCTs; 19 yolk sac tumor, YST) and in the GCT cell lines NCCIT, TE76.T, JAR and 2102Ep, and correlated its expression with AFP and hCG protein levels, histologic differentiation, and clinical follow-up data. EPCAM protein was visualized by immunohistochemistry of selected corresponding paraffin embedded tumor tissues. EPCAM was expressed in malignant but not in benign GCTs irrespective of age, sex, site and clinical stage of tumor (P = 0.001). In primary teratomas, EPCAM expression increased with their grade of immaturity (mean 2(-ΔCt) values: MT 0.23, IT 1.61, P = 0.007) and significantly correlated with serum AFP (P = 0.03) and hCG (P = 0.03) levels in malignant GCTs. Particularly high EPCAM levels were found in nonseminomatous GCTs such as YSTs (8.49) and choriocarcinoma (13.54). Immunohistochemical analysis verified gene expression data showing a distinct EPCAM staining in YST. Similarly in vitro, highest EPCAM expression was measured in GCT cell lines comprising yolk sac (2102Ep: 5.59) or choriocarcinoma (JAR: 10.65) components. This first comprehensive analysis of EPCAM in GCTs revealed high EPCAM expression in YSTs and choriocarcinomas. Thus, these nonseminomatous GCTs may be interesting targets for EPCAM immunotherapy, which has to be evaluated in further studies.

Next-generation-sequencing-based risk stratification and identification of new genes involved in structural and sequence variations in near haploid lymphoblastic leukemia.

Near haploidy (23-29 chromosomes) is a numerical cytogenetic aberration in childhood acute lymphoblastic leukemia (ALL) associated with particularly poor outcome. In contrast, high hyperdiploidy (51-67 chromosomes) has a favorable prognosis. Correct classification and appropriate risk stratification of near haploidy is frequently hampered by the presence of apparently high hyperdiploid clones that arise by endoreduplication of the original near haploid clone. We evaluated next-generation-sequencing (NGS) to distinguish between "high hyperdiploid" leukemic clones of near haploid and true high hyperdiploid origin. Five high hyperdiploid ALL cases and the "high hyperdiploid" cell line MHH-CALL-2, derived from a near haploid clone, were tested for uniparental isodisomy. NGS showed that all disomic chromosomes of MHH-CALL-2, but none of the patients, were of uniparental origin, thus reliably discriminating these subtypes. Whole-exome- and whole-genome-sequencing of MHH-CALL-2 revealed homozygous non-synonymous coding mutations predicted to be deleterious for the protein function of 63 genes, among them known cancer-associated genes, such as FANCA, NF1, TCF7L2, CARD11, EP400, histone demethylases, and transferases (KDM6B, KDM1A, PRDM11). Only eight of these were also, but heterozygously, mutated in the high hyperdiploid patients. Structural variations in MHH-CALL-2 include a homozygous deletion (MTAP/CDKN2A/CDKN2B/ANRIL), a homozygous inversion (NCKAP5), and an unbalanced translocation (FAM189A1). Together, the sequence variations provide MHH-CALL-2 with capabilities typically acquired during cancer development, e.g., loss of cell cycle control, enhanced proliferation, lack of DNA repair, cell death evasion, and disturbance of epigenetic gene regulation. Poorer prognosis of near haploid ALL most likely results from full penetrance of a large array of detrimental homozygous mutations.

In vitro-differentiated embryonic stem cells give rise to male gametes that can generate offspring mice.

Male gametes originate from a small population of spermatogonial stem cells (SSCs). These cells are believed to divide infinitely and to support spermatogenesis throughout life in the male. Here, we developed a strategy for the establishment of SSC lines from embryonic stem (ES) cells. These cells are able to undergo meiosis, are able to generate haploid male gametes in vitro, and are functional, as shown by fertilization after intracytoplasmic injection into mouse oocytes. Resulting two-cell embryos were transferred into oviducts, and live mice were born. Six of seven animals developed to adult mice. This is a clear indication that male gametes derived in vitro from ES cells by this strategy are able to induce normal fertilization and development. Our approach provides an accessible in vitro model system for studies of mammalian gametogenesis, as well as for the development of new strategies for the generation of transgenic mice and treatment of infertility.

Analysis of the adenomatous polyposis coli (APC) gene in childhood and adolescent germ cell tumors.

BACKGROUND: Aberrant Wnt signaling due to deregulation of Wnt regulators is implicated in the development and progression of numerous embryonal tumors. This study addresses the questions if activation of Wnt signaling in germ cell tumors (GCTs) arising during childhood and adolescence is associated with aberrations of the tumor suppressor adenomatous polyposis coli (APC), and whether APC aberrations might be responsible for progression from benign teratoma to malignant yolk sac tumor (YST). PROCEDURE: Forty-eight GCTs were analyzed, including mature (n = 5) and immature (n = 7) teratomas, mixed malignant GCTs (n = 10), YSTs (n = 17) as well as dysgerminomas (n = 9). To screen APC for genetic aberrations, we conducted direct sequencing of the mutation cluster region (MCR), loss of heterozygosity analyses (LOH) and protein truncation test. Epigenetic analyses included methylation specific PCR and bisulfite genomic sequencing of the APC 1a promoter. Gene expression was determined by quantitative real-time PCR. RESULTS: Aberrant promoter methylation was detected in YSTs, teratomas and mixed malignant GCTs, with a pronounced hypermethylation exclusively in YSTs (11/13) while dysgerminomas were not methylated (0/9). Teratomas (2/2) and YSTs (4/5) show LOH at the APC locus. However, neither mutations within the MCR nor truncated protein were detected. APC expression did not significantly vary between the different histological subgroups. CONCLUSIONS: Methylation of APC and LOH 5q21-22 in YSTs and teratomas provide evidence for involvement of APC in the accumulation of ß-catenin and activation of the WNT pathway. Our additional analyses suggest that APC is unlikely to be solely responsible for the formation and progression of childhood GCTs.

Mutational and Functional Analysis of FANCB as a Candidate Gene for Sporadic Head and Neck Squamous Cell Carcinomas.

BACKGROUND/AIM: Head and neck squamous cell carcinomas (HNSCCs) form a heterogeneous tumor entity located throughout the oral cavity, pharynx and larynx that is caused predominantly by chemically or virally induced carcinogenesis. Heterozygous germline mutations in cancer susceptibility genes might also lead to increased incidence of HNSCCs. As DNA stability is typically impaired in HNSCC cells and genes of the Fanconi anemia/BRCA DNA repair pathway can be mutated or down-regulated in HNSCCs, we investigated here whether germline mutations occur in the X-chromosomal FANCB as candidate gene. MATERIALS AND METHODS: Germline DNA of 85 consecutive HNSCC patients was sequenced. Missense alterations in FANCB were functionally tested in reference cells. RESULTS AND CONCLUSION: Four single nucleotide polymorphisms were identified, three of which were located in untranslated regions of FANCB (rs2188383, rs2375729, rs2905223) and predicted to be associated with normal function. One missense alteration, c.1004G>A resulting in p.G335E (rs41309679), in exon 4 was detected in five men in homozygous and in five women in heterozygous state. Four in silico prediction programs uniformally predicted p.G335E to be associated with loss-of-function of the protein. To clarify these predictions, we expressed the FANCB p.G335E protein in primary human FANCB deficient fibroblasts. Cell cycle analysis of these fibroblasts established that the FANCB p.G335E was functionally indistinguishable from the wildtype FANCB protein. Thus, functional studies in genetically defined cells showed that the p.G335E germline alteration in FANCB is not associated with impaired function.

Infection Exposure Promotes ETV6-RUNX1 Precursor B-cell Leukemia via Impaired H3K4 Demethylases.

ETV6-RUNX1 is associated with the most common subtype of childhood leukemia. As few ETV6-RUNX1 carriers develop precursor B-cell acute lymphocytic leukemia (pB-ALL), the underlying genetic basis for development of full-blown leukemia remains to be identified, but the appearance of leukemia cases in time-space clusters keeps infection as a potential causal factor. Here, we present in vivo genetic evidence mechanistically connecting preleukemic ETV6-RUNX1 expression in hematopoetic stem cells/precursor cells (HSC/PC) and postnatal infections for human-like pB-ALL. In our model, ETV6-RUNX1 conferred a low risk of developing pB-ALL after exposure to common pathogens, corroborating the low incidence observed in humans. Murine preleukemic ETV6-RUNX1 pro/preB cells showed high Rag1/2 expression, known for human ETV6-RUNX1 pB-ALL. Murine and human ETV6-RUNX1 pB-ALL revealed recurrent genomic alterations, with a relevant proportion affecting genes of the lysine demethylase (KDM) family. KDM5C loss of function resulted in increased levels of H3K4me3, which coprecipitated with RAG2 in a human cell line model, laying the molecular basis for recombination activity. We conclude that alterations of KDM family members represent a disease-driving mechanism and an explanation for RAG off-target cleavage observed in humans. Our results explain the genetic basis for clonal evolution of an ETV6-RUNX1 preleukemic clone to pB-ALL after infection exposure and offer the possibility of novel therapeutic approaches. Cancer Res; 77(16); 4365-77. ©2017 AACR.

Next-generation-sequencing of recurrent childhood high hyperdiploid acute lymphoblastic leukemia reveals mutations typically associated with high risk patients.

20% of children suffering from high hyperdiploid acute lymphoblastic leukemia develop recurrent disease. The molecular mechanisms are largely unknown. Here, we analyzed the genetic landscape of five patients at relapse, who developed recurrent disease without prior high-risk indication using whole-exome- and whole-genome-sequencing. Oncogenic mutations of RAS pathway genes (NRAS, KRAS, FLT3, n=4) and deactivating mutations of major epigenetic regulators (CREBBP, EP300, each n=2 and ARID4B, EZH2, MACROD2, MLL2, each n=1) were prominent in these cases and virtually absent in non-recurrent cases (n=6) or other pediatric acute lymphoblastic leukemia cases (n=18). In relapse nucleotide variations were detected in cell fate determining transcription factors (GLIS1, AKNA). Structural genomic alterations affected genes regulating B-cell development (IKZF1, PBX1, RUNX1). Eleven novel translocations involved the genes ART4, C12orf60, MACROD2, TBL1XR1, LRRN4, KIAA1467, and ELMO1/MIR1200. Typically, patients harbored only single structural variations, except for one patient who displayed massive rearrangements in the context of a germline tumor suppressor TP53 mutation and a Li-Fraumeni syndrome-like family history. Another patient harbored a germline mutation in the DNA repair factor ATM. In summary, the relapse patients of our cohort were characterized by somatic mutations affecting the RAS pathway, epigenetic and developmental programs and germline mutations in DNA repair pathways.