Validation of a 40-Gene Expression Profile Test to Predict Metastatic Risk in Localized High-Risk Cutaneous Squamous Cell Carcinoma

Background: Current staging systems for cutaneous squamous cell carcinoma (cSCC) have limited positive predictive value (PPV) for identifying patients who will experience metastasis. Objective: To develop and validate a gene expression profile (GEP) test for predicting risk for metastasis in localized, high-risk cSCC with the goal of improving risk-directed patient management. Methods: Archival formalin-fixed paraffin-embedded primary cSCC tissue and clinicopathologic data (n=586) were collected from 23 independent centers in a prospectively designed study. A GEP signature was developed using a discovery cohort (n=202) and validated in a separate, non-overlaping, independent cohort (n=324). Results: A prognostic, 40-gene expression profile (40-GEP) test was developed and validated, stratifying high-risk cSCC patients into classes based on metastasis risk: Class 1 (low-risk), Class 2A (high-risk), and Class 2B (highest-risk). For the validation cohort, 3-year metastasis-free survival (MFS) rates were 91.4%, 80.6%, and 44.0%, respectively. A PPV of 60% was achieved for the highest-risk group (Class 2B), an improvement over staging systems; while negative predictive value, sensitivity, and specificity were comparable to staging systems. Limitations: Potential understaging of cases could affect metastasis rate accuracy.Conclusion: The 40-GEP test is an independent predictor of metastatic risk that can complement current staging systems for patients with high-risk cSCC

Mast Cells and Gastrointestinal Dysmotility in the Cystic Fibrosis Mouse

BACKGROUND: Cystic fibrosis (CF) has many effects on the gastrointestinal tract and a common problem in this disease is poor nutrition. In the CF mouse there is an innate immune response with a large influx of mast cells into the muscularis externa of the small intestine and gastrointestinal dysmotility. The aim of this study was to evaluate the potential role of mast cells in gastrointestinal dysmotility using the CF mouse (Cftr(tm1UNC), Cftr knockout). METHODOLOGY: Wild type (WT) and CF mice were treated for 3 weeks with mast cell stabilizing drugs (ketotifen, cromolyn, doxantrazole) or were treated acutely with a mast cell activator (compound 48/80). Gastrointestinal transit was measured using gavage of a fluorescent tracer. RESULTS: In CF mice gastric emptying at 20 min post-gavage did not differ from WT, but was significantly less than in WT at 90 min post-gavage. Gastric emptying was significantly increased in WT mice by doxantrazole, but none of the mast cell stabilizers had any significant effect on gastric emptying in CF mice. Mast cell activation significantly enhanced gastric emptying in WT mice but not in CF mice. Small intestinal transit was significantly less in CF mice as compared to WT. Of the mast cell stabilizers, only doxantrazole significantly affected small intestinal transit in WT mice and none had any effect in CF mice. Mast cell activation resulted in a small but significant increase in small intestinal transit in CF mice but not WT mice. CONCLUSIONS: The results indicate that mast cells are not involved in gastrointestinal dysmotility but their activation can stimulate small intestinal transit in cystic fibrosis

Transit of rhodamine dextran in the small intestine and effect of mast cell agents.

<p>Overnight fasted mice were gavaged with a bolus of rhodamine dextran (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004283#s2" target="_blank">Methods</a>). At 20 min post-gavage the mice were sacrificed and the distribution of the fluorescence in 10 segments (proximal to distal) of the small intestine was measured and expressed relative to the total fluorescence. (A) WT Control (n = 28); (B) CF Control (n = 14); (C) Ketotifen, 3 week treatment of WT (n = 10); (D) Ketotifen, 3 week treatment of CF (n = 6); (E) Cromolyn, 3 week treatment of WT (n = 11); (F) Cromolyn, 3 week treatment of CF (n = 7); (G) Doxantrazole, 3 week treatment of WT (n = 5); (H) Doxantrazole, 3 week treatment of CF (n = 8); (I) Compound 48/80, acute i.p. injection of WT (n = 4); (J) Compound 48/80, acute i.p. injection of CF (n = 6). Data are means ± SEM. (*) <i>P</i><0.05 vs control, same genotype and same intestinal segment; (+) <i>P</i><0.05 CF vs WT, same segment and treatment group. Data for the control WT and CF mice include values from previous work <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004283#pone.0004283-DeLisle1" target="_blank">[1]</a>.</p

Gastric emptying in WT and CF mice and effect of mast cell agents.

<p>Overnight fasted mice were gavaged with a bolus of rhodamine dextran (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004283#s2" target="_blank">Methods</a>). At 20 min post-gavage the mice were sacrificed and the distribution of the fluorescence in the stomach and small intestine was measured. Gastric emptying is expressed as fraction of total fluorescence which has exited the stomach in the 20 min post-gavage period. Con = control (n = 28 WT, n = 14 CF); Ket = ketotifen, 3 week treatment (n = 10 WT, n = 6 CF); Crom = cromolyn 3 week treatment (n = 11 WT, n = 7 CF); Dox = doxantrazole 3 week treatment (n = 5 WT , n = 8 CF); 48/80 = compound 48/80 acute i.p. injection (n = 4 WT, n = 6 CF). Data are means ± SEM. (*) <i>P</i><0.05 vs Control of the same genotype. Data for the control WT and CF mice include values from previous work <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004283#pone.0004283-DeLisle1" target="_blank">[1]</a>.</p

Distribution of rhodamine dextran along the gastrointestinal tract of WT and CF mice at 20 min and 90 min post-gavage.

<p>Overnight fasted mice were gavaged with a bolus of rhodamine dextran (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004283#s2" target="_blank">Methods</a>). At either 20 min or 90 min post-gavage the mice were sacrificed and the distribution of the fluorescence in the stomach (St), small intestine (SI) and large intestine (LI) was measured and expressed relative to the total. (A) WT mice (n = 28 mice 20 min post-gavage, n = 8 mice 90 min post-gavage); (B) CF mice (n = 14 mice 20 min post-gavage, n = 7 mice 90 min post-gavage). Data are means ± SEM. (*) <i>P</i><0.05 comparing 20 min vs 90 min data for the same organ and genotype of mice. (+) <i>P</i><0.05 comparing CF vs WT for the same organ and post-gavage time.</p

Next-Generation Bisulfite Sequencing of Aml Reveals Widespread Acquisition of Epigenetic Abnormalities in Leukemic Stem Cells That Are Stably Retained in More Mature Leukemic Cell Fractions

Abstract Acute myeloid leukemia (AML) is a disease of aberrant hematopoietic differentiation believed to mirror the hierarchical pattern of hematopoiesis with leukemia stem cells (LSC) serving as the originating cell population from which the tumor arises. Like hematopoietic stem cells (HSC), leukemia stem cells are believed to be largely quiescent and therefore impervious to conventional chemotherapeutics resulting in relapse of disease despite achievement of clinical remission. The DNA methylation profiles of bulk leukemia cells differ significantly from normal CD34+ cells; however, less is known about the potential differences between epigenetic profiles of purified LSC and normal HSC. Moreover, the stability of the methylome as the LSC differentiate into mature leukemia progenitor cells (LPC) has not been studied. In order to address this LSC, LPC, and HSC were sorted from the bone marrow of AML patients and normal controls based on CD34, CD38, CD45 and ALDH activity. LSC were defined as CD34+ CD38- ALDHmid; LPC as CD34+ CD38+; and HSC as CD34+ CD38- ALDHhigh. These isolated fractions were used for genome-wide DNA methylation analysis by the next-generation enhanced reduced representation bisulfite sequencing (ERRBS) assay, which allowed for the comparison of the methylation landscapes of LSC and HSC, as well as those of LSC and LPC. A total of thirteen AML samples were examined for the presence of LSC, six of which did not have an ALDHmid population but had instead an ALDHhigh population. Because of their phenotypic similarity to normal HSC, these samples were not included in the present comparison against HSC. The methylomes of six independent LSC samples were compared to methylomes of five HSC. Sequenced ERRBS libraries were aligned against the human genome (hg19) and differentially methylated regions (DMR) were identified using a beta-binomial model and selecting regions with absolute mean methylation difference of >25% and false discovery rate (FDR) < 10%. The methylation profiles of LSC showed widespread genome wide differences relative to HSC; 39,162 regions were found to be hypermethylated in LSC while 5,408 regions were hypomethylated. DMRs were enriched at CpG islands as well as intra- and intergenic enhancers. Functional annotation of the DMRs to gene sets in the MSigDb database revealed enrichment for genes marked by the Polycomb repressive mark H3K27me3 (FDR = 1.86×10-49). In order to determine whether epigenetic abnormalities observed at the LSC level were distinct from the epigenetic profiles observed in the more mature LPC fraction, we compared paired LSC and LPC specimens from 6 AML patients. Notably, LPC did not significantly differ in their epigenetic profiles from LSC, indicating that epigenetic abnormalities acquired at the LSC stage are stably transmitted through leukemic expansion to the more mature LPC fraction. In summary, we have identified widespread epigenetic abnormalities acquired at the LSC stage, of greater magnitude than was previously recognized by performing comparisons of leukemic cells to unfractionated CD34+ controls. Genes targeted by aberrant methylation in LSC are significantly enriched in Polycomb target genes, suggesting a potential role for Polycomb proteins in leukemic transformation. By contrast, no significant epigenetic differences were observed between the LSC and LPC fractions in AML, indicating that epigenetic abnormalities acquired at the LSC level are static through multiple cell generations. Disclosures Gore: Celgene: Consultancy, Honoraria, Research Funding

Aldehyde Dehydrogenase Activity in the Leukemic Stem Cell Compartment Uncovers Opposing Methylation Patterns of Leukemia Stem Cells in AML

Abstract Acute myeloid leukemia (AML) is a disease marked by abnormal differentiation of the myeloid cell lineage. Leukemia stem cells differentiate to give rise to leukemic progenitor cells (LPC) and ultimately leukemic blasts which are not leukemia-initiating. Previous studies have revealed a diverse methylation landscape in AML, but have mostly relied on the blast population rather than more purified primitive populations. Consequently, the status of the leukemic methylome during expansion from LSC to LPC and finally blast remains largely unknown. We sorted HSC from the bone marrow of normal donors and LSC, LPC, and blasts from AML patients based on expression of CD34, CD38, CD45 and aldehyde dehydrogenase (ALDH) activity. Normal HSC were defined as CD34+CD38-ALDHhigh. In AML patients, LSC were either CD34+CD38-ALDHhigh or CD34+CD38-ALDHmid; LPC were CD34+CD38+; the blast population consisted of unsorted mononuclear cells. Though patients with an ALDHhigh LSC profile may have had some residual normal HSCs present, contribution of these cells was likely minimal and thus the overall population predominantly leukemic. Methylation profiles for each cell fraction in eight untreated AML patients and five normal donors were generated using the enhanced reduced representation bisulfite sequencing (ERRBS) assay. All sequenced ERRBS libraries were aligned against the human genome (hg19) and organized into 25 base pair tiles for analysis of differentially methylated regions (DMR) using a beta-binomial model that takes variation across samples into account during DMR identification. DMR classification required a difference in methylation of >25% and false discovery rate (FDR) < 10%. Unsupervised correspondence analysis indicated that methylomes of two patients with ALDHhigh LSC were distinct from the six patients with ALDHmid LSC and therefore patients were grouped based on the ADLH activity of their LSCs for comparisons. Both ALDHhigh LSC and ALDHmid LSC had extensive alterations in methylation across their genomes when compared to HSC. The great majority of DMRs in ALDHhigh LSC were hypomethylated; of the 62,415 DMRs identified, 55,418 regions were hypomethylated while only 6,997 were hypermethylated in ALDHhigh LSC. In contrast, in ALDHmid LSC, 39,162 DMRs were hypermethylated and 5,408 regions hypomethylated compared to HSC. Despite opposing patterns of methylation, DMRs were enriched at intergenic and intragenic enhancers in both ALDHhigh and ALDHmid LSC. DMRs were functionally annotated to gene sets in the MSigDb database. Genes associated with ALDHhigh DMRs were enriched for genes with the binding motif for transcription factor Sp1 near their promoters (FDR = 2.55×10-79) and ALDHmid DMR associated genes were enriched for genes with H3K27 trimethylation in their promoters (FDR = 7.19×10-169). We compared methylation profiles of LSC to LPC and blasts in an effort to determine whether changes to the methylome occur with leukemic maturation. Interestingly, there were no significant changes in methylation between LSC and LPC in either ALDH population. However, we did see changes to the epigenome emerge when LSC or LPC were compared to leukemic blasts. In ALDHhigh patients, LSC and LPC were more hypomethylated than blasts while ALDHmid LSC and LPC were more hypermethylated than the more differentiated blasts. In conclusion, alterations to the LSC methylome were extensive and two patterns of methylation emerged based on the ALDH activity of LSC; ALDHhigh LSC displayed hypomethylated profiles and ALDHmid LSC were hypermethylated. Enrichment of DMRs at intra and intergenic enhancer regions in both LSC types despite their opposing methylation patterns highlights the importance of epigenetic marks in these regions and their role as regulators of gene expression. Significant changes in methylation between LSC or LPC and blasts, but not LSC and LPC suggest relative stability of the methylome during early leukemic differentiation with more substantial alterations occurring after the LPC level. Disclosures Gerber: Janssen: Research Funding; Alexion: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Carraway:Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; Baxalta: Speakers Bureau. Gore:Celgene: Consultancy, Honoraria, Research Funding