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

Clinical Performance and Management Outcomes with the DecisionDx-UM Gene Expression Profile Test in a Prospective Multicenter Study

Uveal melanoma management is challenging due to its metastatic propensity. DecisionDx-UM is a prospectively validated molecular test that interrogates primary tumor biology to provide objective information about metastatic potential that can be used in determining appropriate patient care. To evaluate the continued clinical validity and utility of DecisionDx-UM, beginning March 2010, 70 patients were enrolled in a prospective, multicenter, IRB-approved study to document patient management differences and clinical outcomes associated with low-risk Class 1 and high-risk Class 2 results indicated by DecisionDx-UM testing. Thirty-seven patients in the prospective study were Class 1 and 33 were Class 2. Class 1 patients had 100% 3-year metastasis-free survival compared to 63% for Class 2 (log rank test p=0.003) with 27.3 median follow-up months in this interim analysis. Class 2 patients received significantly higher-intensity monitoring and more oncology/clinical trial referrals compared to Class 1 patients (Fisher’s exact test p=2.1×10-13 and p=0.04, resp.). The results of this study provide additional, prospective evidence in an independent cohort of patients that Class 1 and Class 2 patients are managed according to the differential metastatic risk indicated by DecisionDx-UM. The trial is registered with Clinical Application of DecisionDx-UM Gene Expression Assay Results (NCT02376920)

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

Distinct DNA Methylation and Expression Profiles Underlie CMML Responsiveness to Decitabine and Uncover Novel Mechanism of Resistance

Abstract Myelodysplastic syndromes (MDS) and the related disorder chronic myelomonocytic leukemia (CMML) are characterized by abnormal DNA hypermethylation and the DNA methyltransferase inhibitors (DMTis) azacytidine and decitabine (DAC) are frequently used as frontline therapy in these patients. However, DMTis are ineffective for ~50% of the patients who must still undergo treatment for at least 6 months before they can be deemed resistant. Therefore, it is of critical importance to identify baseline molecular differences associated with DMTi sensitivity that can help (i) to improve patient risk-stratification at diagnosis and (ii) identify the underlying mechanisms of resistance to these agents. Previous efforts to identify baseline DNA methylation differences at promoter regions between DMTi responders and non-responders have not been successful, so we hypothesized that any potential differences would be located distally from promoter regions. For this purpose we studied 40 CMML patients at diagnosis, all of whom had been uniformly treated with DAC 20 mg/m2/day x 5 days as frontline therapy. After 6 cycles of therapy patients were classified as responders (n=19, hematological improvement or better), or non-responders (n=21, stable or progressive disease). Mutational analysis showed no significant differences in the frequency of mutations in TET2, ASXL1, DNMT3A, RUNX1, TP53, JAK2, KIT, KRAS, EZH2, IDH1/2 and spliceosome genes. Using Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) we analyzed the baseline methylation status at ~3M CpG sites across the genome of 39/40 CMML patients. We identified 158 statistically significant differentially methylated regions (DMRs) (FDR<0.1 and methylation difference ≥25%) between the two groups. DAC-sensitive patients displayed both regions of higher methylation as well as regions with lower methylation compared to DAC-resistant patients. As predicted, DMRs were depleted at promoters (DMRs 9% vs. Background [BG] 21%, p-value: 3.4×10-5) and CpG islands (DMRs 8% vs. BG 25%, p-value: 1.5×10-8). Further analysis showed that hypermethylated DMRs were enriched at intronic regions (Hyper DMRs 58% vs. BG 33%, p-value: 3.7×10-6) while hypomethylated DMRs were enriched at intergenic regions (Hypo DMRs 49% vs. BG 38%, p-value: 2.6×10-2). Moreover, hypermethylated DMRs were significantly enriched for enhancer regions, and in particular, enhancers located within gene bodies (hyper DMRs 38% vs. BG 18%, p-value: 2.3×10-5). KEGG pathway analysis showed a significant enrichment of DMRs in the MAPK signaling pathway (FDR<0.01). Next, using a support vector machine algorithm with 10-fold cross validation we were able to develop a classifier capable of predicting response to DAC with high level of accuracy (ROC AUC: 0.99) based solely on the DNA methylation status at diagnosis of 17 genomic regions. Three different random splits of the cohort into training and test sets achieved correct predictions for 85.7%, 89.47%, and 100% of cases, respectively, demonstrating the accuracy and potential utility of such a classifier. Finally, RNA-seq analysis identified 53 differentially expressed genes between responders (n=8) and non-responders (n=6) at diagnosis. Genes implicated in cell cycle and DNA replication were overexpressed in responders. By contrast, very few genes were overexpressed at the time of diagnosis in primary resistant patients. Among these were CXCL4 and CXCL7 which, given their reported contributions to cell cycle arrest and chemoresistance, were tested for their functional roles in DAC resistance. Pre-treatment of normal CD34+ cells for 72 h with 10nM DAC significantly reduced colony formation (p<0.05) but the addition of 50ng/mL of CXCL4 and CXCL7 restored colony formation to that of untreated cells. Moreover, treatment of primary CMML cells with 10 nM DAC for 72h significantly reduced viability of these cells, while concomitant treatment with 50ng/mL of CXCL4 and CXCL7 was sufficient to abrogate this effect. Taken together, our findings demonstrate that (i) specific DNA methylation profiles targeting non-promoter regulatory regions are associated with DAC sensitivity, (ii) these differences can be harnessed for the development of clinical biomarkers predictive of response and (iii) we identified a novel mechanism of resistance to DAC mediated through two chemokines that are exclusively overexpressed in non-responders. Disclosures No relevant conflicts of interest to declare

Molecular Determinants of Decitabine Response in Chronic Myelomonocytic Leukemia

Abstract The nucleoside analog (na) 5-aza-2’-deoxycytidine (dac) has good, but heterogeneous, efficacy in the therapy of chronic myelomonocytic leukemia (cmml). Given that no standard therapy has been identified so far for cmml, there is urgent need to identify molecular markers that could support the choice of dac therapy and identify patients more prone to respond. Recently, we demonstrated that in mds patients the expression of uck1, involved in the activation of azacitidine, may influence the clinical response to this treatment (valencia et al, leukemia 2013). In the present study, we assessed the role of dac metabolizing enzymes as well as genetic alterations common to cmml patients in response to dac in a uniformly and prospectively treated cohort of cmml patients. Methods: Patients Forty cmml patients were treated with dac 20mg/m2/day for 5 days every 28 d. DNA and rna were extracted from bm mononuclear cells of 19 pts defined as responders to dac (r), and 21 as non-responders (nr). Hematological response was evaluated according to iwg 2006 criteria. Gene mutation. the fifteen most frequently mutated genes in cmml were sequenced at a mean depth of coverage of 520x (range 169–714x). Functional studies. the role of two main genes involved in dac metabolism: dck (dac activation) and dctd (dac deactivation) was evaluated by silencing both genes with sirnas. The experiments were performed in the mds-skm1 cell line and in primary cells of 6 cmml cases exposed in vitro to dac 10um for 48h. Gene expression. the expression level of genes hent1, hent2, dctd, hcnt3, cn-ii, dck and cda, involved in dac metabolism was evaluated by qrt-pcr in 38/40 cmml patients and by rnaseq in 14/40 patients. CDNA libraries were sequenced using the hiseq 2000. The counts of endogenous genes were normalized by ercc spike-in library controls, and differential expression analysis was performed using edger (v3.4.2) glm model. The differentially expressed genes were identified at the fdr cutoff of 0.05 and absolute fold change greater than 2. Results: in skm-1 cells and in cmml primary mononuclear cells, dctd silencing increased dac- induced apoptosis (annexin v-positive cells 20.2%±0.8% vs control 13.8%±0.5%; p=0.01). dck silencing led to a decrease in dac-induced apoptosis (annexin v-positive cells 8.8%±0.1% vs control 13.8%±0.5%; p=0.05). We could not detect by rnaseq a significant difference in the expression levels of na metabolizing enzymes between responders and non responders to dac. qrt-pcr confirmed these observations. The mutational frequencies (figure) in this cohort of cmml cases were: srsf2 50%, tet2 44.7%, asxl1 39.4%, nras 18.4%, runx1 and dnmt3a 10.5%, u2af1 and tp53 7.8%, kras, jak2 and kit 5.2%, ezh2, idh1, idh2 and sf3b1 2.6%. No single genetic alteration was significantly associated with shorter overall survival or resistance to dac. Conclusions: although we could clearly demonstrate that in vitro expression of dac metabolizing enzymes influenced response to dac, clinical resistance does not appear to be directly correlated with the expression of genes involved in dac metabolism nor to specific gene mutations and is likely determined by other clinical/molecular variables that remain to be identified. Figure 1 Figure 1. Disclosures Finelli: Janssen: Speaker, Speaker Other; Novartis: Speaker, Speaker Other; Celgene: Research Funding, Speaker Other

Specific molecular signatures predict decitabine response in chronic myelomonocytic leukemia

Myelodysplastic syndromes and chronic myelomonocytic leukemia (CMML) are characterized by mutations in genes encoding epigenetic modifiers and aberrant DNA methylation. DNA methyltransferase inhibitors (DMTis) are used to treat these disorders, but response is highly variable, with few means to predict which patients will benefit. Here, we examined baseline differences in mutations, DNA methylation, and gene expression in 40 CMML patients who were responsive or resistant to decitabine (DAC) in order to develop a molecular means of predicting response at diagnosis. While somatic mutations did not differentiate responders from nonresponders, we identified 167 differentially methylated regions (DMRs) of DNA at baseline that distinguished responders from nonresponders using next-generation sequencing. These DMRs were primarily localized to nonpromoter regions and overlapped with distal regulatory enhancers. Using the methylation profiles, we developed an epigenetic classifier that accurately predicted DAC response at the time of diagnosis. Transcriptional analysis revealed differences in gene expression at diagnosis between responders and nonresponders. In responders, the upregulated genes included those that are associated with the cell cycle, potentially contributing to effective DAC incorporation. Treatment with CXCL4 and CXCL7, which were overexpressed in nonresponders, blocked DAC effects in isolated normal CD34(+) and primary CMML cells, suggesting that their upregulation contributes to primary DAC resistance

Lettre à Monsieur des Alliers d'Argenville, Maître des Comptes, de l'Académie Royale des Sciences de Montpellier, au sujet d'un tableau appartenant au Roi, et dont il parle dans son ouvrage de la vie des Peintres / [Chappotin de Saint-Laurent, Michel]

Référence bibliographique : Deloynes, 122

Mutation allele burden remains unchanged in chronic myelomonocytic leukaemia responding to hypomethylating agents

International audienceThe cytidine analogues azacytidine and 5-aza-2'-deoxycytidine (decitabine) are commonly used to treat myelodysplastic syndromes, with or without a myeloproliferative component. It remains unclear whether the response to these hypomethylating agents results from a cytotoxic or an epigenetic effect. In this study, we address this question in chronic myelomonocytic leukaemia. We describe a comprehensive analysis of the mutational landscape of these tumours, combining whole-exome and whole-genome sequencing. We identify an average of 14±5 somatic mutations in coding sequences of sorted monocyte DNA and the signatures of three mutational processes. Serial sequencing demonstrates that the response to hypomethylating agents is associated with changes in DNA methylation and gene expression, without any decrease in the mutation allele burden, nor prevention of new genetic alteration occurence. Our findings indicate that cytosine analogues restore a balanced haematopoiesis without decreasing the size of the mutated clone, arguing for a predominantly epigenetic effect