Expanded analysis of secondary germline findings from matched tumor/normal sequencing identifies additional clinically significant mutations.

BACKGROUND: Next-generation sequencing (NGS) for tumor molecular profiling can reveal secondary germline pathogenic and likely pathogenic variants (LPV/PV). The American College of Medical Genetics (ACMG) recommends return of secondary results for a subset of 59 genes, but other genes with evidence of clinical utility are emerging. We previously reported that 4.3% of patients who underwent NGS of a targeted panel of 201 genes had LPV/PV based on the ACMG list. Here we report the frequency of additional germline cancer-related gene variants and discuss their clinical utility. PATIENTS AND METHODS: Matched tumor and germline DNA NGS of a targeted panel of 201 genes was performed in a research laboratory on samples from 1000 patients with advanced or metastatic solid tumors enrolled in a molecular testing protocol (NCT01772771). The frequency of germline LPV/PV in 54 cancer-related genes, beyond the genes in ACMG list, were analyzed. RESULTS: Among 1000 patients who underwent tumor/normal DNA sequencing, 46 (4.6%) were found to have a germline LPV/PV in the following genes: AR-(5), ATM-(4), BAP1-(1), CDH1-(1), CDKN2A-(1), CHEK1-(2), CHEK2-(10), EGFR-(1), ERCC3-(4), ERCC5-(1), HNF1B-(1), HRAS-(1), MITF-(4), MLL3-(1), NF1-(3), PKHD1-(4), PTCH1-(1), and SMARCA4-(1). Thus, a total 8.7% of patients had an LPV/PV with 2 patients having 2 concomitant germline LPV/PV. Five mutations in high-penetrance hereditary cancer predisposition genes were selected to be returned to patients or their representatives: BAP1, CDH1, CDKN2A, EGFR, and SMARCA4. CONCLUSIONS: Broader genomic testing is likely to identify additional secondary pathogenic germline alterations, some with potential clinical utility for return to patients and their relatives. The recommended genes for which germline results should be returned are continually changing, warranting continued study.

Incidental germline variants in 1000 advanced cancers on a prospective somatic genomic profiling protocol.

BACKGROUND: Next-generation sequencing in cancer research may reveal germline variants of clinical significance. We report patient preferences for return of results and the prevalence of incidental pathogenic germline variants (PGVs). PATIENTS AND METHODS: Targeted exome sequencing of 202 genes was carried out in 1000 advanced cancers using tumor and normal DNA in a research laboratory. Pathogenic variants in 18 genes, recommended for return by The American College of Medical Genetics and Genomics, as well as PALB2, were considered actionable. Patient preferences of return of incidental germline results were collected. Return of results was initiated with genetic counseling and repeat CLIA testing. RESULTS: Of the 1000 patients who underwent sequencing, 43 had likely PGVs: APC (1), BRCA1 (11), BRCA2 (10), TP53 (10), MSH2 (1), MSH6 (4), PALB2 (2), PTEN (2), TSC2 (1), and RB1 (1). Twenty (47%) of 43 variants were previously known based on clinical genetic testing. Of the 1167 patients who consented for a germline testing protocol, 1157 (99%) desired to be informed of incidental results. Twenty-three previously unrecognized mutations identified in the research environment were confirmed with an orthogonal CLIA platform. All patients approached decided to proceed with formal genetic counseling; in all cases where formal genetic testing was carried out, the germline variant of concern validated with clinical genetic testing. CONCLUSIONS: In this series, 2.3% patients had previously unrecognized pathogenic germline mutations in 19 cancer-related genes. Thus, genomic sequencing must be accompanied by a plan for return of germline results, in partnership with genetic counseling.

Clinical massively parallel next-generation sequencing analysis of 409 cancer-related genes for mutations and copy number variations in solid tumours.

BACKGROUND: In a clinical diagnostic laboratory, we evaluated the applicability of the Ion Proton sequencer for screening 409 cancer-related genes in solid tumours. METHODS: DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue biopsy specimens of 55 solid tumours (20 with matched normal tissue) and four cell lines and screened for mutations in 409 genes using the Ion Proton system. The mutation profiles of these samples were known based on prior testing using the Ion Torrent Personal Genome Machine (46-gene hotspot panel), Sanger sequencing, or fluorescence in situ hybridisation (FISH). Concordance with retrospective findings and additional mutations were evaluated. Assay sensitivity and reproducibility were established. Gene copy number variations (CNVs) detected were confirmed by molecular inversion probe (MIP) array. RESULTS: The average Ion Proton (409-gene panel) sequencing output per run was 8 gigabases with 128 million sequencing reads. Of the 15,992 amplicons in the 409-gene panel, 90% achieved a minimum average sequencing depth of 100X. In 59 samples, the Ion Proton detected 100 of 105 expected single-nucleotide variants (SNVs) and all expected deletions (n=8), insertions (n=5), and CNVs (n=7). Five SNVs were not detected due to failed amplification of targeted regions. In 20 tumours with paired normal tissue, Ion Proton detected 37 additional somatic mutations, several in genes of high prognostic or therapeutic significance, such as MET, ALK, TP53, APC, and PTEN. MIP array analysis confirmed all CNVs detected by Ion Proton. CONCLUSIONS: The Ion Proton (409-gene panel) system was found to be well suited for use in a clinical molecular diagnostic laboratory. It can simultaneously screen 409 genes for a variety of sequence variants in multiple samples using a low input of FFPE DNA with high reproducibility and sensitivity.

Seizures, cell death, and mossy fiber sprouting in kainic acid-treated organotypic hippocampal cultures.

Sprouting of the mossy fiber axons of the dentate granule cells is a structural neuronal plasticity found in the mature brain of epileptic humans and experimental animals. Mossy fiber sprouting typically arises in experimental animals after repeated seizures and may contribute to the hyperexcitability of the epileptic brain. Investigation of the molecular triggers and spatial cues involved in mossy fiber sprouting has been hampered by the lack of an optimal in vitro model for studying this rearrangement. For an in vitro model to be feasible, the circuitry and receptors involved in convulsant-induced mossy fiber sprouting would have to be localized near the granule cells, rather than being dependent on long-range brain interconnections. However, it is not known whether this is the case. We report here that that application of the convulsant, kainic acid, to organotypic hippocampal explant cultures induces seizures, neuronal cell death, and subsequent dramatic mossy fiber sprouting with a similar laminar preference and time-course to that seen in intact animals. Prolonged (48 h) but not transient (4 h) kainic acid treatment caused regionally selective neuronal cell death. Cultures treated with kainic acid for a prolonged period displayed a time- and dose-dependent increase in supragranular Timm staining reflective of increased mossy fiber innervation to this area. Direct visualization of mossy fiber axons with neurobiotin-labeling revealed that mossy fibers in kainic acid-treated cultures exhibited a dramatic increase in supragranular axonal branch points and synaptic boutons. The cellular and molecular determinants required for kainic acid-induced cell death and subsequent mossy fiber reorganization thus appear to be intrinsic to the hippocampal slice preparation, and are preserved in culture. Given the ease with which functional inhibitors or pharmacological agents may be utilized in this system, slice cultures may provide a powerful model in which to study the molecular components involved in triggering mossy fiber outgrowth and underlying its laminar specificity. Elucidation of these molecular pathways will likely have both specific utility in clarifying the functional consequences of mossy fiber sprouting, as well as general utility in understanding of synaptic reorganization in the mature central nervous system.

Immunohistochemical evidence of seizure-induced activation of trk receptors in the mossy fiber pathway of adult rat hippocampus.

Recent work suggests that limiting the activation of the trkB subtype of neurotrophin receptor inhibits epileptogenesis, but whether or where neurotrophin receptor activation occurs during epileptogenesis is unclear. Because the activation of trk receptors involves the phosphorylation of specific tyrosine residues, the availability of antibodies that selectively recognize the phosphorylated form of trk receptors permits a histochemical assessment of trk receptor activation. In this study the anatomy and time course of trk receptor activation during epileptogenesis were assessed with immunohistochemistry, using a phospho-specific trk antibody. In contrast to the low level of phosphotrk immunoreactivity constitutively expressed in the hippocampus of adult rats, a striking induction of phosphotrk immunoreactivity was evident in the distribution of the mossy fibers after partial kindling or kainate-induced seizures. The anatomic distribution, time course, and threshold for seizure-induced phosphotrk immunoreactivity correspond to the demonstrated pattern of regulation of BDNF expression by seizure activity. These results provide immunohistochemical evidence that trk receptors undergo activation during epileptogenesis and suggest that the mossy fiber pathway is particularly important in the pro-epileptogenic effects of the neurotrophins.

Selective inhibition of kindling development by intraventricular administration of TrkB receptor body.

Recent work has shown that neurotrophin gene expression is increased after seizures evoked in the kindling model of epilepsy, but whether neurotrophins regulate kindling development is as yet unclear. In this study, we attempted to block selectively the activation of distinct neurotrophin receptors throughout kindling development in the rat via chronic intracerebroventricular administration of trk receptor bodies. The efficacy and selectivity of the trk receptor bodies were established by inhibition of neurotrophin-induced trk receptor phosphorylation in pheochromocytoma (PC12) cells and primary cultures of cortical neurons. The intracerebroventricular infusion of trkB receptor body (trkB-Fc) inhibited development of kindling in comparison with that seen with saline or human IgG controls, trkA-Fc, or trkC-Fc. These results imply that activation of trkB receptors contributes to the development of kindling, a form of activity-dependent behavioral plasticity in the adult mammalian brain.

Neurovirulence determinants of genetically engineered Theiler viruses.

Theiler murine encephalomyelitis viruses (TMEVs) are picornaviruses that cause enteric and neurological disease in mice. The GDVII strain and other members of the GDVII subgroup are highly virulent and cause an acute, fatal polioencephalomyelitis following intracerebral inoculation, whereas the DA stain and other members of the TO subgroup cause a persistent, demyelinating infection. We previously produced a full-length, infectious DA cDNA clone. We now describe the generation of a full-length, infectious GDVII cDNA clone and the subsequent production of intratypic chimeric cDNAs and intratypic recombinant viruses. Inoculation of the recombinant viruses into mice demonstrated that a major determinant of TMEV neurovirulence is within the GDVII 1B (capsid protein VP2)-2C coding region, most likely in the GDVII 1B (VP2)-2A coding region. Genomic sequences 5' to this region of GDVII RNA also contribute to expression of the full neurovirulence phenotype. These data demonstrate the multigenic nature of TMEV neurovirulence, as has been reported for other viruses.

Polyclonal IgM anti-GM1 ganglioside antibody in patients with motor neuron disease and variants.

Recent studies reported the presence of anti-ganglioside antibodies in occasional patients with motor neuron disease. We found polyclonal serum IgM anti-GM1 antibodies by an anti-GM1 enzyme-linked immunosorbent assay (ELISA) in 9 (19%) of 48 patients with motor neuron disease. A comparable frequency of IgM anti-GM1 antibodies was found in 4 (10%) of 40 sera from patients with other neurological disease. Three (17%) of 18 sera from the patients with motor neuron disease and 2 (17%) of 12 sera from patients with other neurological diseases had anti-GM1 immunostaining as shown by thin layer chromatography immunoblot. One patient with a lower motor neuron variant of motor neuron disease or motor axonopathy without multifocal conduction block had a markedly elevated polyclonal IgM anti-GM1 ELISA titer (greater than 1:64,000) with prominent immunostaining of GM1, moderate immunostaining of GM2, and weak and inconsistent immunostaining of GD1b by thin layer chromatography immunoblot. Treatment with prednisone resulted in clinical improvement despite increasing anti-GM1 antibody titers. These data indicate that patients with motor neuron disease have measurable levels of anti-ganglioside antibodies as frequently as patients with other neurological diseases. This contrasts with a small subgroup of patients with a lower motor neuron variant of motor neuron disease or motor axonopathy who have markedly elevated levels of serum anti-ganglioside antibodies and a clinical syndrome that is treatable with immunosuppression.

Theiler's murine encephalomyelitis virus neutralization escape mutants have a change in disease phenotype.

DA strain of Theiler's murine encephalomyelitis virus produces a persistent demyelinating infection. We previously produced escape mutant viruses that are resistant to a neutralizing monoclonal antibody and have a mutation in VP1 amino acid residue 268 in a neutralization site (Y. Ohara, A. Senkowski, J. Fu, L. Klaman, J. Goodall, M. Toth, and R.P. Roos, J. Virol. 62:3527-3529, 1988). In contrast to wild-type DA strain, these escape mutants produce little if any demyelinating disease after inoculation into weanling mice.