A Highly Sensitive Pan-Cancer Test for Microsatellite Instability.

Microsatellite instability (MSI) is an evolving biomarker for cancer detection and treatment. MSI was first used to identify patients with Lynch syndrome, a hereditary form of colorectal cancer (CRC), but has recently become indispensable in predicting patient response to immunotherapy. To address the need for pan-cancer MSI detection, a new multiplex assay was developed that uses novel long mononucleotide repeat (LMR) markers to improve sensitivity. A total of 469 tumor samples from 20 different cancer types, including 319 from patients with Lynch syndrome, were tested for MSI using the new LMR MSI Analysis System. Results were validated by using deficient mismatch repair (dMMR) status according to immunohistochemistry as the reference standard and compared versus the Promega pentaplex MSI panel. The sensitivity of the LMR panel for detection of dMMR status by immunohistochemistry was 99% for CRC and 96% for non-CRC. The overall percent agreement between the LMR and Promega pentaplex panels was 99% for CRC and 89% for non-CRC tumors. An increased number of unstable markers and the larger size shifts observed in dMMR tumors using the LMR panel increased confidence in MSI determinations. The LMR MSI Analysis System expands the spectrum of cancer types in which MSI can be accurately detected

Leukemogenesis in heterozygous PU.1 knockout mice

Most murine radiation-induced acute myeloid leukemias involve biallelic inactivation of the PU.1 gene, with one allele being lost through a radiation-induced chromosomal deletion and the other allele affected by a recurrent point mutation in codon 235 that is likely to be spontaneous. The short latencies of acute myeloid leukemias occurring in nonirradiated mice engineered with PU.1 conditional knockout or knockdown alleles suggest that once both copies of PU.1 have been lost any other steps involved in leukemogenesis occur rapidly. Yet, spontaneous acute myeloid leukemias have not been reported in mice heterozygous for a PU.1 knockout allele, an observation that conflicts with the understanding that the PU.1 codon 235 mutation is spontaneous. Here we describe experiments that show that the lack of spontaneous leukemia in PU.1 heterozygous knockout mice is not due to insufficient monitoring times or mouse numbers or the genetic background of the knockout mice. The results reveal that spontaneous leukemias that develop in mice of the mixed 129S2/SvPas and C57BL/6 background of knockout mice arise by a pathway that does not involve biallelic PU.1 mutation. In addition, the latency of radiation-induced leukemia in PU.1 heterozygous mice on a genetic background susceptible to radiation-induced leukemia indicates that the codon 235 mutation is not a rate-limiting step in radiation leukemogenesis driven by PU.1 loss

<i>Sleeping Beauty</i> Mouse Models Identify Candidate Genes Involved in Gliomagenesis

<div><p>Genomic studies of human high-grade gliomas have discovered known and candidate tumor drivers. Studies in both cell culture and mouse models have complemented these approaches and have identified additional genes and processes important for gliomagenesis. Previously, we found that mobilization of <i>Sleeping Beauty</i> transposons in mice ubiquitously throughout the body from the <i>Rosa26</i> locus led to gliomagenesis with low penetrance. Here we report the characterization of mice in which transposons are mobilized in the Glial Fibrillary Acidic Protein (GFAP) compartment. Glioma formation in these mice did not occur on an otherwise wild-type genetic background, but rare gliomas were observed when mobilization occurred in a <i>p19Arf</i> heterozygous background. Through cloning insertions from additional gliomas generated by transposon mobilization in the <i>Rosa26</i> compartment, several candidate glioma genes were identified. Comparisons to genetic, epigenetic and mRNA expression data from human gliomas implicates several of these genes as tumor suppressor genes and oncogenes in human glioblastoma.</p></div

Genotypes and phenotypes of analyzed brains from GFAP-SB11 crosses.

a<p>Abbreviations used: GFAP-SB = GFAP-SB11 transposase, T2 = T2/onc, p19 = p19Arf, wt = wild-type.</p><p>Genotypes and phenotypes of analyzed brains from GFAP-SB11 crosses.</p

The mutational, copy number, methylation and mRNA expression status of gCIS/CIS human orthologs in TCGA GBM data.

a<p>For <i>Kpna4/Gm1647</i> data presented are for <i>KPNA4</i> as <i>Gm1647</i> does not have a human ortholog.</p>b<p>Abbreviations used: N/A = not applicable (no human ortholog, or data not available), ns = non-significant, fc = fold change.</p>c<p>The percentage of tumors harboring hypermethylation and evidence for decreased mRNA expression (epigenetic silencing) are shown in parentheses.</p><p>The mutational, copy number, methylation and mRNA expression status of gCIS/CIS human orthologs in TCGA GBM data.</p

Genotypes and phenotypes of analyzed brains from Rosa26-SB11 crosses.

a<p>Abbreviations used: T2LC = T2/onc LC, T2ATG = T2/oncATG, p19 = p19Arf, wt = wild-type, AA = anaplastic astrocytoma, GBM = glioblastoma, PNET = primitive neuroectodermal tumor, DD = differential diagnosis.</p><p>Genotypes and phenotypes of analyzed brains from Rosa26-SB11 crosses.</p

FLI1 is expressed in a subset of cells in a glioma with a <i>Fli1</i> insertion.

<p>Immunoreactivity for FLI1 is in brown and immunoreactivity for IBA1 is in green. Nuclei are counterstained blue. A) 40× image of the dorsal third ventricular region in a control mouse without mobilizing transposons. B) 100× image of the boxed area in A. Asterisk indicates a FLI1 immunoreactive cell with morphologic features of a red blood cell. C) 40× image of the dorsal third ventricular region surrounded by tumor in AR151. D) 100× image of the boxed area in C. Arrowhead points to a nucleus that is negative for FLI1 and an arrow indicates an example of strong nuclear FLI1 staining. E) 100× image of tumor in AR151 that is distant from the ventricle. F) A 40× image of secondary only controls is shown for comparison to verify specific primary antibody staining. Scale bars = 50 µm.</p