SUMMARY To characterize patient-derived xenografts (PDXs) for functional studies, we made whole-genome comparisons with originating breast cancers representative of the major intrinsic subtypes. Structural and copy number aberrations were found to be retained with high fidelity. However, at the single-nucleotide level, variable numbers of PDX-specific somatic events were documented, although they were only rarely functionally significant. Variant allele frequencies were often preserved in the PDXs, demonstrating that clonal representation can be transplantable. Estrogen-receptor-positive PDXs were associated with ESR1 ligand-binding-domain mutations, gene amplification, or an ESR1/YAP1 translocation. These events produced different endocrine-therapy-response phenotypes in human, cell line, and PDX endocrine-response studies. Hence, deeply sequenced PDX models are an important resource for the search for genome-forward treatment options and capture endocrine-drug-resistance etiologies that are not observed in standard cell lines. The originating tumor genome provides a benchmark for assessing genetic drift and clonal representation after transplantation.

Methylation of the CpG-rich region (CpG island) overlapping a gene’s promoter is a generally accepted mechanism for silencing expression. While recent technological advances have enabled measurement of DNA methylation and expression changes genome-wide, only modest correlations between differential methylation at gene promoters and expression have been found. We hypothesize that stronger associations are not observed because existing analysis methods oversimplify their representation of the data and do not capture the diversity of existing methylation patterns. Recently, other patterns such as CpG island shore methylation and long partially hypomethylated domains have also been linked with gene silencing. Here, we detail a new approach for discovering differential methylation patterns associated with expression change using genome-wide high-resolution methylation data: we represent differential methylation as an interpolated curve, or signature, and then identify groups of genes with similarly shaped signatures and corresponding expression changes. Our technique uncovers a diverse set of patterns that are conserved across embryonic stem cell and cancer data sets. Overall, we find strong associations between these methylation patterns and expression. We further show that an extension of our method also outperforms other approaches by generating a longer list of genes with higher quality associations between differential methylation and expression.

We have examined the acute effects of insulin and isoproterenol on the phosphorylation state of the insulinregulatable glucose transporter (IRGT) in rat adipocytes. The IRGT was immunoprecipitated from either detergentsolubilized whole-cell homogenates or subcellular fractions of 32P-labeled fat cells and subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The 32P-labeled IRGT was detected by autoradiography as a species ofapparent Mr 46,000. Insulin stimulated translocation of the IRGT from low-density microsomes to the plasma membrane but did not affect phosphorylation of the transporter in either fraction. Isoproterenol inhibited insulin-stimulated glucose transport by 40% but was without effect on the subcellular distribution of the transporter in either the presence or absence of insulin.Isoproterenol stimulated phosphorylation of the IRGT 2-fold. Incubating cells with dibutyryl-cAMP and 8-bromo-cAMP also stimulated phosphorylation 2-fold, and the transporter was phosphorylated in vitro when IRGT-enriched vesicles were incubated with cAMP-dependent protein kinase and [Y-32P]ATP. These results suggest that isoproterenol stimulates phosphorylation of the IRGT via a cAMP-dependent pathway and that phosphorylation of the transporter may modulate its ability to transport glucose.

In lung adenocarcinoma, canonical EML4-ALK inversion results in a fusion protein with a constitutively active ALK kinase domain. Evidence of ALK rearrangement occurs in a minority (2-7%) of lung adenocarcinoma, and only ~60% of these patients will respond to targeted ALK inhibition by drugs such as crizotinib and ceritinib. Clinically, targeted anti-ALK therapy is often initiated based on evidence of an ALK genomic rearrangement detected by fluorescence in situ hybridization (FISH) of interphase cells in formalin-fixed, paraffin-embedded tissue sections. At the genomic level, however, ALK rearrangements are heterogeneous, with multiple potential breakpoints in EML4, and alternate fusion partners. Using next-generation sequencing of DNA and RNA together with ALK immunohistochemistry, we comprehensively characterized genomic breakpoints in 33 FISH-positive lung adenocarcinomas. Of these 33 cases, 29 (88%) had detectable DNA level ALK rearrangements involving EML4, KIF5B, or non-canonical partners including ASXL2, ATP6V1B1, PRKAR1A, and SPDYA. A subset of 12 cases had material available for RNA-Seq. Of these, eight of eight (100%) cases with DNA rearrangements showed ALK fusion transcripts from RNA-Seq; three of four cases (75%) without detectable DNA rearrangements were similarly negative by RNA-Seq, and one case was positive by RNA-Seq but negative by DNA next-generation sequencing. By immunohistochemistry, 17 of 19 (89%) tested cases were clearly positive for ALK protein expression; the remaining cases had no detectable DNA level rearrangement or had a non-canonical rearrangement not predicted to form a fusion protein. Survival analysis of patients treated with targeted ALK inhibitors demonstrates a significant difference in mean survival between patients with next-generation sequencing confirmed EML4-ALK rearrangements, and those without (20.6 months vs 5.4 months, P<0.01). Together, these data demonstrate abundant genomic heterogeneity among ALK-rearranged lung adenocarcinoma, which may account for differences in treatment response with targeted ALK inhibitors.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia caused by the expansion of a hematopoietic progenitor cell that has acquired a mutation in the X-linked PIGA gene. PNH occurs on the background of bone marrow failure. Bone marrow failure and the presence of the abnormal cells account for the clinical phenotype of patients with PNH including hemolysis, cytopenia, and thrombophilia. PIGA is essential for the synthesis of glycosyl phosphatidylinositol (GPI) anchor molecules. PNH blood cells are therefore deficient in all proteins that use such an anchor molecule for attachment to the cell membrane. Two of these proteins regulate complement activation on the cell surface. Their deficiency therefore explains the exquisite sensitivity of PNH red blood cells to complement-mediated lysis. Complement-mediated lysis of red blood cells is intravascular, and intravascular hemolysis contributes significantly to the morbidity and mortality in patients with this condition. PNH is an outstanding example of how an increased understanding of pathophysiology may directly improve the diagnosis, care, and treatment of disease.