Clinical and biological effects of demethylating agents on solid tumours – a systematic review Accepted Manuscript Systematic or Meta analysis Studies Clinical and biological effects of demeth[.]
Accepted Manuscript Systematic or Meta-analysis Studies Clinical and biological effects of demethylating agents on solid tumours – a systematic review J.F Linnekamp, R Butter, R Spijker, J.P Medema, H.W.M van Laarhoven PII: DOI: Reference: S0305-7372(17)30004-X http://dx.doi.org/10.1016/j.ctrv.2017.01.004 YCTRV 1595 To appear in: Cancer Treatment Reviews Cancer Treatment Reviews Received Date: Revised Date: Accepted Date: November 2016 January 2017 January 2017 Please cite this article as: Linnekamp, J.F., Butter, R., Spijker, R., Medema, J.P., van Laarhoven, H.W.M., Clinical and biological effects of demethylating agents on solid tumours – a systematic review, Cancer Treatment Reviews Cancer Treatment Reviews (2017), doi: http://dx.doi.org/10.1016/j.ctrv.2017.01.004 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain Article type: Systematic review Clinical and biological effects of demethylating agents on solid tumours – a systematic review J F Linnekampa, R Buttera, R Spijkerb, J P Medemaa, H W M van Laarhovena,c Affiliation list: a Laboratory of Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands; Cancer Genomics Center, Amsterdam, The Netherlands b Cochrane Netherlands, Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, University Utrecht, Utrecht, The Netherlands; Medical Library, Academic Medical Center (AMC), University of Amsterdam, Meibergdreef 9,1105 AZ Amsterdam, The Netherlands c Department of Medical Oncology, Academic Medical Center (AMC), University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands Correspondence to: professor Hanneke W M van Laarhoven, MD, PhD, PhD, Department of Medical Oncology, Academic Medical Centre, Meibergdreef 9; F4-224, 1105 AZ Amsterdam, the Netherlands, telephone number +31 20 56 65955, email address: h.vanlaarhoven@amc.uva.nl Clinical and biological effects of demethylating agents on solid tumours – a systematic review Abstract Background: It is assumed that DNA methylation plays a key role in both tumour development and therapy resistance Demethylating agents have been shown to be effective in the treatment of haematological malignancies Based on encouraging preclinical results, demethylating agents may also be effective in solid tumours This systematic review summarizes the evidence of the effect of demethylating agents on clinical response, methylation and the immune system in solid tumours Methods: We conducted a systematic literature search from 1949 to December 2016, according to the PRISMA guidelines Studies which evaluated treatment with azacitidine, decitabine, guadecitabine, hydralazine, procaine, MG98 and/or zebularine in patients with solid tumours were included Data on clinical response, effects on methylation and immune response were extracted Results: Fifty eight studies were included: in 13 studies complete responses (CR) were observed, 35 studies showed partial responses (PR), 47 studies stable disease (SD) and all studies except two showed progressive disease (PD) Effects on global methylation were observed in 11/15 studies and demethylation/re-expression of tumour specific genes was seen in 15/17 studies No clear correlation between (de)methylation and clinical response was observed In 14 studies immune-related responses were reported, such as reexpression of cancer-testis antigens and upregulation of interferon genes Conclusion: Demethylating agents are able to improve clinical outcome and alter methylation status in patients with solid tumours Although beneficial effect has been shown in individual patients, overall response is limited Further research on biomarker predicting therapy efficacy is indicated, particularly in earlier stage and highly methylated tumours Keywords: Epigenetics, Solid tumour, Demethylating agents, DNMT inhibitor, Therapy resistance, Immune response Introduction Epigenetics is the process of heritable changes of the chromosome without changing the DNA sequence itself [1] Distinctive processes that drive alterations of the epigenome include DNA methylation, RNA associated silencing and histone (de)acetylation [1] Epigenetic changes such as global hypomethylation, particularly at centrometric repeats, as well as hypermethylation, often occurring in CpG islands, are frequently present in cancer [2] Hypomethylation has been linked to genomic instability, while CpG island hypermethylation is affecting gene expression by altering the chromatin structure [3] In cancer, genes affected by hypermethylation are involved in apoptosis, cell cycle control, DNA repair and drug metabolism among others [4-6] Furthermore, DNA methylation in solid tumours has been associated with therapy resistance and poor prognosis [4, 7] Hence, hypermethylation is an attractive target for treatment in order to influence tumour biology and potentially to overcome therapy resistance Methylation of DNA is catalysed by a group of enzymes called DNA methyltransferases (DNMTs) [8] While DNMT1 is active during maintenance of methylated cytosine during DNA replication thus copying the methylome to the progeny, DNMT3 is responsible for de novo methylation at previous unmethylated sites [8, 9] In contrast to mutations, methylation must be actively maintained and is thereby potentially reversible Two classes of demethylating agents are available: nucleoside DNMT inhibitors (DNMTi) and non-nucleoside DNMT inhibitors In the early sixties, two nucleoside DNMT inhibitors were discovered, namely 5azacytidine (azacitidine, AZA) and its derivate 5-2’-deoxycytidine (decitabine, DAC) Although several preclinical and clinical trials in the sixties and seventies were performed that showed promising effects mainly on leukaemia in both mouse models [10] and patients [11], it took until 1980 to discover that azacitidine and decitabine were able to influence cellular differentiation by demethylation [12] Inhibition of DNMT is causing a reduction in overall DNA methylation rather than demethylation of a specific (target) gene In the last four decades these agents have been examined for anti-cancer activity, mostly in haematological malignancies Clinical trials have shown that demethylation is more effective when demethylating agents are administered at low concentrations, thereby also limiting dose dependent toxicity [13] Recently, other nucleoside DNMT inhibitors, zebularine and guadecitabine and nonnucleoside DNMT inhibitors, such as hydralazine, procaine, and MG98 have been identified and are currently being investigated as demethylating drugs Today, only azacitidine and decitabine are approved by the US Food and Drug Administration (FDA) for treatment in MDS [14, 15] In order to determine effects on methylation of these drugs, a great arsenal of assays is available and can be used for various clinical or research purposes Briefly, the methylation assays can be divide in three groups: single-CpG resolution assays, global methylation assays and relative DNA methylation assays comparing samples to a suitable reference [16] Single-CpG resolution assays provide an absolute methylation and include amplicon bisulphite sequencing and pyrosequencing among others Global methylation assays measure the total DNA methylation content of a sample making it popular for assessing the effect of demethylating agents In contrast, relative DNA methylation assays like Methylight make use of a reference sample with predefined DNA methylation, usually with fully methylated DNA [16] In a recent comparison amplicon bisulphite sequencing and bisulphite pyrosequencing showed the best all-round performance [16] Alternatively, for (re-) expression of genes/protein RT-PCR, microarray and western blot can be used Although this approach is not measuring methylation nor demethylation, it allows for the assessment of downstream effect on gene/protein expression Thus far, demethylating agents are not approved for the treatment of solid tumours However, preclinical studies in solid tumours have shown that over-expression of DNMTs is associated with resistance to chemotherapy [17, 18] Also, targets for therapy, such as the EGF receptor, can be silenced by methylation, which makes colon cancer patients resistant to cetuximab [19] Furthermore, several preclinical studies in solid tumours have indicated that treatment with DNMTi can reverse chemoresistance, including resistance to platinumderived agents [20], induce apoptosis [21] and target cancer stem cells [22] Although the effect of demethylating agents on methylation is irrefutable, the correlation between demethylation and response is less evident Therefore also other biological effects of demethylating agents should be considered and examined Modification of the immune system is a potential new function for demethylating agents Upregulation of immune related genes in cancer cells by demethylating agents can lead to an enhanced recognition by Tcells [23] For instance the activation of cancer–testis antigens (CTA) like MAGE and NYESO1 after treatment with demethylating agents has been described in several tumour types [24-26] Preclinical studies suggest that treatment with decitabine can mimic a viral infection by induction of dsRNAs [27, 28] Furthermore, the ability of demethylating agents to activate the immune system, makes the combination with drugs such as checkpoint inhibitors or vaccine based treatment a potential strategy [29] In this systematic review, we summarizes the evidence on clinical response and biological effects of demethylating agents in solid tumours We also examine whether there are patient characteristics that are associated with improved response to demethylating agents Finally, we will describe the relation between demethylating agents and effects on the immune system based on the available studies Methods We conducted a systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines [30] Literature search We conducted a systematic literature search using EMBASE, PubMed and Web of Science up to December 6th, 2016 The query was developed and executed in PubMed and subsequently translated to other databases No additional filters were used The query as used in PubMed is presented in box Reference lists of retrieved articles were screened for additional hits Box PubMed query ((("Azacitidine"[Mesh] OR "Azacitidine"[tiab] OR "5 AZC"[tiab] OR "Azacytidine"[tiab] OR "decitabine" [Supplementary Concept] OR "decitabine"[tiab] OR "5-aza-2'-deoxycytidine"[tiab] OR "Dezocitidine"[tiab] OR "DAC"[tiab] OR "zebularine"[tiab] OR "pyrimidin-2-one beta-ribofuranoside"[Supplementary Concept] OR "pyrimidin-2-one beta-ribofuranoside"[tiab] OR "1-beta-D-ribofuranosyl-2(1H)-pyrimidinone"[tiab] OR "Pyrimidin-2one beta-D-ribofuranoside"[tiab] OR "Pyrimidin-2-one ribonucleoside"[tiab] OR "1-beta-D-ribofuranosylpyrimidin2(1H)-one"[tiab] OR "DHZ"[tiab] OR "Procaine"[Mesh] OR "Procaine"[tiab] OR "2-Diethylaminoethyl paminobenzoate"[tiab] OR "p-Aminobenzoic acid 2-diethylaminoethyl ester"[tiab] OR "Vitamin H3"[tiab] OR "beta(diethylamino)ethyl 4-aminobenzoate"[tiab] OR "beta-(diethylamino)ethyl p-aminobenzoate"[tiab] OR "Hydralazine"[Mesh] OR "Hydralazine"[tiab]) OR (MG98[tiab] OR MG-98[tiab] OR SGI-110[tiab] OR s110[tiab] OR ("MG 98 phosphorothioate antisense oligodeoxynucleotide"[Supplementary Concept]) OR "S110 compound "[Supplementary Concept])) AND ("Neoplasms"[Mesh] OR "cancer"[tiab] OR "malignancy"[tiab] OR "malignancies"[tiab] OR sarcoma*[tiab] OR tumour[tiab] OR tumour[tiab] OR tumours[tiab] OR tumours[tiab] OR cancer[tiab] OR neoplasm*[tiab]) AND (((clinical[Title/Abstract] AND trial[Title/Abstract]) OR clinical trials[MeSH Terms] OR clinical trial[Publication Type] OR random*[Title/Abstract] OR random allocation[MeSH Terms] OR therapeutic use[MeSH Subheading]) OR (Epidemiologic studies[mesh] OR case control studies[mesh] OR cohort studies[mesh] OR Case control[tw] OR cohort study[tw] OR cohort studies[tw] OR Cohort analy*[tw] OR Follow up study[tw] OR follow up studies[tw] OR (observational study[tw] OR observational studies[tw] OR Longitudinal[tw] OR Retrospective[tw] OR Cross sectional[tw] OR Cross-sectional studies[mesh])))) NOT (animals[mesh] NOT humans[mesh]) Study selection Randomized controlled trials (RCTs), cohort studies and case reports in English studying adults of all ethnicities from both genders with any type of solid tumour were considered eligible Intervention criteria included treatment with azacitidine, decitabine, guadecitabine, hydralazine, MG98, procaine or zebularine, either as a single agent or administered in combination with other agents/treatment Studies reporting on clinical response mainly based on radiological assessment (complete response (CR), partial response (PR), progressive disease (PD), stable disease (SD)) and (de)methylation measurements in blood or tumour tissue were selected Data extraction and quality assessment JFL and RB independently screened title and abstract and if applicable the full articles Duplicates were removed Discrepancies were resolved by discussion between both reviewers until consensus was reached The following primary information was collected: name of the first author, year of publication, country of the study, phase of the study, number of included patients, clinical/radiological outcome (CR, PD, PR and SD) and data on methylation and the immune system in blood or tumour samples Studies were rated independently by both reviewers using an adapted version of the Newcastle-Ottawa quality assessment Scale (NOS) cohort studies [31] Studies with the highest ranking were described individually Synthesis We described detailed characteristics and the main findings of the included RCTs, cohort studies and the case report, as reported by the authors of the included studies Secondly, we described the effect of demethylating agents on methylation, gene expression and the immune system Results Study characteristics A total of 3610 publication records were obtained through a search in electronic databases (Figure 1) After removal of duplicates, 2991 unique publications were screened based on title and abstract Both clinical trials and pre-clinical trials were present as well as studies describing both We decided for such a broad search to ensure that all clinical studies of interest were obtained and to avoid exclusion of studies describing both pre-clinical as well as clinical results Full texts of 135 publications were screened of which 58 studies met our inclusion criteria Of these 58 studies 53 were cohort studies, two were RCTs, two included methylation data of included cohort studies and one was a case report A detailed overview of the included studies is available in Table Methodological quality of included studies To assess the quality of the 58 included studies, studies were systematically rated using an scoring system based on the Newcastle-Ottawa Scale (NOS) for cohort studies (Supplemental Figure 1) Of the 55 studies that reported clinical outcomes, 37 presented validated outcome measures (WHO or RECIST criteria), while the other 18 studies described outcome by a self-generated measure In three studies, a description of the outcome was incomplete, as stable disease was not mentioned [32-34] In three studies, clinical response was not an outcome measure and only results on methylation/demethylation were shown [35-37] Scoring of the studies was done as follows: for the inclusion of control groups 20 points could be obtained, for all other criteria 10 points were given The methodological quality of the included studies ranged from 30 to 80% for the cohort studies and 90 and 100% for the two RCTs The studies with the highest quality were a double blind RCT [38] and a non-blinded RCT [39] (Table 2) Clinical and biological outcomes Studies that were included described a large variety of solid tumours The majority (33/58) studied one specific type of cancer, e.g melanoma, ovarian cancer, breast cancer and cervical cancer The remaining 25 studies included more than one type of solid tumour (Table 3) The majority of the studies (46/58) used azacitidine or decitabine (both 23 studies), five studies used hydralazine and four studies used MG98 Demethylating drugs were most frequently used in combination with other anti-cancer therapy, mainly conventional [17] Wang C, Mirkin BL, Dwivedi RS DNA (cytosine) methyltransferase overexpression is associated with acquired drug resistance of murine neuroblastoma cells International journal of oncology 2001;18(2):323-9 [18] Segura-Pacheco B, Perez-Cardenas E, Taja-Chayeb L, Chavez-Blanco A, Revilla-Vazquez A, Benitez-Bribiesca L, et al Global DNA hypermethylation-associated cancer chemotherapy resistance and its reversion with the demethylating agent hydralazine Journal of translational medicine 2006;4:32 [19] Scartozzi M, Bearzi I, Mandolesi A, Giampieri R, Faloppi L, Galizia E, et al Epidermal growth factor receptor (EGFR) gene promoter methylation and cetuximab treatment in colorectal cancer patients Br J Cancer 2011;104(11):1786-90 [20] Qiu YY, Mirkin BL, Dwivedi RS Inhibition of DNA methyltransferase reverses cisplatin induced drug resistance in murine neuroblastoma cells Cancer detection and prevention 2005;29(5):456-63 [21] Soengas MS, Capodieci P, Polsky D, Mora J, Esteller M, Opitz-Araya X, et al Inactivation of the apoptosis effector Apaf-1 in malignant melanoma Nature 2001;409(6817):207-11 [22] Liu L, Chen L, Wu X, Li X, Song Y, Mei Q, et al Low-dose DNA-demethylating agent enhances the chemosensitivity of cancer cells by targeting cancer stem cells via the upregulation of microRNA-497 Journal of cancer research and clinical oncology 2016;142(7):1431-9 [23] Klar AS, Gopinadh J, Kleber S, Wadle A, Renner C Treatment with 5-Aza-2'-Deoxycytidine Induces Expression of NY-ESO-1 and Facilitates Cytotoxic T Lymphocyte-Mediated Tumor Cell Killing PloS one 2015;10(10):e0139221 [24] De Smet C, De Backer O, Faraoni I, Lurquin C, Brasseur F, Boon T The activation of human gene MAGE-1 in tumor cells is correlated with genome-wide demethylation Proceedings of the National Academy of Sciences of the United States of America 1996;93(14):7149-53 [25] Weiser TS, Guo ZS, Ohnmacht GA, Parkhurst ML, Tong-On P, Marincola FM, et al Sequential 5Aza-2 deoxycytidine-depsipeptide FR901228 treatment induces apoptosis preferentially in cancer cells and facilitates their recognition by cytolytic T lymphocytes specific for NY-ESO-1 Journal of immunotherapy (Hagerstown, Md : 1997) 2001;24(2):151-61 [26] Weiser TS, Ohnmacht GA, Guo ZS, Fischette MR, Chen GA, Hong JA, et al Induction of MAGE-3 expression in lung and esophageal cancer cells The Annals of thoracic surgery 2001;71(1):295-301; discussion -2 [27] Roulois D, Loo Yau H, Singhania R, Wang Y, Danesh A, Shen SY, et al DNA-Demethylating Agents Target Colorectal Cancer Cells by Inducing Viral Mimicry by Endogenous Transcripts Cell 2015;162(5):961-73 [28] Chiappinelli KB, Strissel PL, Desrichard A, Li H, Henke C, Akman B, et al Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses Cell 2016;164(5):1073 [29] Coral S 5-aza-2 -Deoxycytidine-induced Expression of Functional Cancer Testis Antigens in Human Renal Cell Carcinoma: Immunotherapeutic Implications 2002;8:2690–5 [30] Liberati A, Altman DG, Tetzlaff J, Mulrow C, Gotzsche PC, Ioannidis JP, et al The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate healthcare interventions: explanation and elaboration BMJ (Clinical research ed) 2009;339:b2700 [31] Available from: http://www.ohri.ca/programs/clinical_epidemiology/oxford.asp [32] Aparicio A, Eads CA, Leong LA, Laird PW, Newman EM, Synold TW, et al Phase I trial of continuous infusion 5-aza-2'-deoxycytidine Cancer Chemother Pharmacol 2003;51(3):231-9 [33] Bellet RE, Mastrangelo MJ, Engstrom PF, Strawitz JG, Weiss AJ, Yarbro JW Clinical trial with subcutaneously administered 5-azacytidine (NSC-102816) Cancer chemotherapy reports 1974;58(2):217-22 [34] Chan AT, Tao Q, Robertson KD, Flinn IW, Mann RB, Klencke B, et al Azacitidine induces demethylation of the Epstein-Barr virus genome in tumors J Clin Oncol 2004;22(8):1373-81 [35] de la Cruz-Hernandez E, Perez-Cardenas E, Contreras-Paredes A, Cantu D, Mohar A, Lizano M, et al The effects of DNA methylation and histone deacetylase inhibitors on human papillomavirus early gene expression in cervical cancer, an in vitro and clinical study Virol J 2007;4:18 19 ...Article type: Systematic review Clinical and biological effects of demethylating agents on solid tumours – a systematic review J F Linnekampa, R Buttera, R Spijkerb, J P Medemaa, H W M van Laarhovena,c... Clinical and biological effects of demethylating agents on solid tumours – a systematic review Abstract Background: It is assumed that DNA methylation plays a key role in both tumour development and. .. resolution assays, global methylation assays and relative DNA methylation assays comparing samples to a suitable reference [16] Single-CpG resolution assays provide an absolute methylation and include