George Dranitsaris

CpG methylation analysis of the MEG3 and SNRPN imprinted genes in acute myeloid leukemia and myelodysplastic syndromes

Methylation is now established as a fundamental regulator of gene transcription. To investigate this in haematologic malignancies, we evaluated the aberrant promoter methylation of two imprinted genes (MEG3 and SNRPN) in 43 MDS and 42 AML patients. MEG3 hypermethylation occurred in 15 MDS patients (34.9%), and in 20 AML patients (47.6%). SNRPN hypermethylation was observed in 15 MDS patients (34.9%), and in 21 AML patients (50%). There were no significant correlations between WHO subtype, WPSS score, karyotype, haemoglobin levels, white blood cell count, platelet count and CpG methylation of any gene. MEG3 hypermethylation was associated with significantly reduced overall survival in individuals with AML (HR=1.98, p=0.04), while SNRPN CpG methylation was not associated with survival (HR=0.94, p=0.87). In addition, no association between survival and aberrant MEG3 (HR=2.15, p=0.072) or SNRPN methylation (HR=1.08, p=0.85) was observed in patients MDS. Our findings suggest that these genes are abnormally methylated in AML and MDS patients, and methylation of MEG3 confers worse overall prognosis. The MEG3 methylation status may serve as a useful biomarker in leukemia.

The prolyl-hydroxylase EGLN3 and not EGLN1 is inactivated by methylation in plasma cell neoplasia

EGLN1 and EGLN3 are members of the egg‐laying‐defective 9 (EglN) prolyl‐hydroxylases which during normoxia catalyse hydroxylation of the hypoxia‐inducible factor (HIF)‐1α, thereby promoting its ubiquitination by a complex containing the von Hippel–Lindau (VHL) tumour suppressor. EGLN3 also has pro‐apoptotic activity in some cell types. Analyses of a well‐characterised series of cases of plasma cell dyscrasias, including multiple myeloma (MM), Waldenström’s macroglobulinaemia (WM) and monoclonal gammopathy of undetermined significance (MGUS) surprisingly demonstrated that the CpG island of EGLN3, and not EGLN1, is frequently methylated in these disorders. Multiple myeloma patients with a methylated EGLN3 promoter showed trends towards an increased risk of death, bone lytic lesions, anaemia, advanced stage of disease and the presence of extramedullary disease. Those individuals with methylation in the EGLN3 CpG island also had significantly lower albumin levels. These data suggest that the prolyl‐hydroxylases may be a novel class of potential tumour suppressors in plasma cell neoplasia that warrant further investigation with regard to their potential utility as biomarkers. Moreover, we observed that EGLN3 is also methylated at high frequency in B‐cell lymphoma subtypes, implying that loss of EGLN3 is an important epigenetic event not only in plasma cell neoplasias but also in B‐cell neoplasias.

Clinical trial design in biosimilar drug development.

SummaryIn contrast to most drugs which are chemically synthesized and have a known structure, biological drugs are derived from living organisms or their products. Biologicals are structurally more complex and unique from chemically synthesized small drug molecules because of their larger size and intricate manufacturing process. Secondary to their protein structure, they are also more prone to acute and chronic immune responses. Biosimilars are intended to offer comparable safety and efficacy relative to reference brand biologicals, yet they are not generic alternatives to the original compounds and so are currently not considered interchangeable. Given their structural complexity, multifaceted manufacturing processes and risk for immunogenicity, biosimilars require class-specific regulatory approval pathways. Here we seek to provide a general overview of clinical trial design in the era of biosimilar drug development. This will include a review of the regulatory requirements for clinical trials in Europe and the United States, followed by a review of two biosimilars that have recently reported results of randomized trials against branded biologicals.

Study of specific genetic and epigenetic variables in multiple myeloma

Few studies have examined the association between methylenetetrahydrofolate reductase (MTHFR) SNPs, epigenetic changes, and multiple myeloma (MM). We wished to determine genotype distributions for MTHFR 1298AC SNP in cases of MM and healthy controls and to examine whether there is any correlation between the methylation status of the CpG island of CDKN2A and Snk/Plk2 and MTHFR genotypes and with overall survival (OS) and other relevant clinical parameters. Bone marrow and peripheral blood were obtained from 45 patients with MM and 77 controls, respectively. The frequencies of the MTHFR 1298AA, 1298AC, and 1298CC genotypes were 53.3%, 40%, and 6.7% for the patient population and 50.6%, 41.6%, and 7.8% for the controls. No statistically significant difference was found in genotype distribution between cases and controls. No correlation was noted between MTHFR genotypes and OS, disease stage, bone disease, anemia, and extramedullary disease. Regarding CDKN2A and Snk/Plk2 CpG island methylation analysis, we found 12 of 45 patients and 27 of 45, respectively, to be methylated. CDKN2A and Snk/Plk2 methylation did not correlate with MTHFR genotypes. Herein, we report the identification of Snk/Plk2 as a novel methylated gene in MM and show that methylation is not influenced in this CpG island or in that of a previously described methylated gene, CDKN2A, in MM. Further evaluation in a larger sample of patients is needed in order to better define the prognostic and clinical value, if any, of MTHFR 1298 polymorphisms and CDKN2A and Snk/Plk2 methylation in the pathogenesis of MM.

von Hippel–Lindau Methylation Status in Patients with Multiple Myeloma: A Potential Predictive Factor for the Development of Bone Disease

It has been suggested that during multiple myeloma (MM) progression, a proangiogenesis stress response occurs, but the mechanistic basis of this has not been established. It is an attractive hypothesis that loss of expression of the von Hippel-Lindau (VHL) gene, resulting in constitutive activation of hypoxia-inducible factor-1alpha (HIF-1alpha), contributes to increased angiogenesis in MM. Because aberrant methylation in the VHL CpG island could cause downregulation of VHL transcription, we prospectively studied the methylation status of the VHL CpG island in 45 individuals with multiple myeloma (MM; 25 men, 20 women; mean age, 66.4 years) and in 10 individuals with borderline thrombocytopenia, who were proven to have no malignancy and served as controls. Methylation was found in 15 of 45 patients with MM at diagnosis (33.3%). The presence of methylation in the VHL CpG island was significantly associated with the development of bone disease (odds ratio, 7.5; P = .018). Patients with bone disease had an increased risk of death compared with those with no bone lytic lesions (hazard ratio [HR], 5.1; P = .13). VHL methylation was not a predictor of excess mortality (HR, 0.92; P = .91). Our data imply that methylation in the VHL CpG island is a frequent event in patients with MM and might be a potential biomarker of bone disease. Methylation in the VHL CpG island, leading to transcriptional silencing and hence decreased HIF-1alpha proteolysis, could be a possible mechanism of increased angiogenesis and altered bone marrow microenvironment that is more supportive for survival and growth of MM cells, contributing to MM bone disease. Whether it represents an early or late event of the disease merits additional study. Additional studies regarding the serum levels of HIF-1alpha and vascular endothelial growth factor would be mechanistically interesting.

Polo-like kinase 2 ( SNK/PLK2 ) is a novel epigenetically regulated gene in acute myeloid leukemia and myelodysplastic syndromes: genetic and epigenetic interactions

Polo-like kinase 2 (SNK/PLK2), a transcriptional target for wild-type p53 and is hypermethylated in a high percentage of multiple myeloma and B cell lymphomas patients. Given these data, we sought to study the methylation status of the specific gene in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), and to correlate it with clinical and genetic features. Using methylation-specific PCR MSP, we analyzed the methylation profile of 45 cases of AML and 43 cases of MDS. We also studied the distribution of MTHFR A1298C and MTHFR C677T polymorphisms and FLT3 mutations in AML patients and correlated the results with hypermethylation in the SNK/PLK2 CpG island. The SNK/PLK2 CpG island was hypermethylated in 68.9% and 88.4% of AML and MDS cases, respectively. Cases with hypermethylation had a trend towards more favorable overall survival (OS). There was no association between different MTHFR genotypes and susceptibility to develop AML. SNK/PLK2 hypermethylation combined with the MTHFR AA1298 genotype was associated with a tendency for a better OS. Similarly, patients with SNK/PLK2 hypermethylation combined with the MTHFR CT677 polymorphism had a better OS (HRu2009=u20090.34; pu2009=u20090.017). SNK/PLK2 methylation associated with unmutated FLT3 cases had a trend for better OS compared to patients with mutated FLT3 gene. SNK/PLK2 is a novel epigenetically regulated gene in AML and MDS, and methylation occurs at high frequency in both diseases. As such, SNK/PLK2 could represent a potential pathogenetic factor, although additional studies are necessary to verify its exact role in disease pathogenesis.

Interpreting results from oncology clinical trials: a comparison of denosumab to zoledronic acid for the prevention of skeletal-related events in cancer patients

BackgroundCritically reviewing the design, endpoints, and results of clinical trials can be challenging to health care professionals. This paper will review the basic methods of presenting clinical outcomes in randomized trials and will focus on the number needed to treat (NNT) concept. NNT will then be applied to the case of bone-targeted therapies denosumab and zoledronic acid, which are used for the prevention of skeletal-related events (SREs) in a variety of disease sites.MethodsA Medline search was performed to identify randomized trials comparing denosumab to zoledronic acid for the prevention of SREs in patients with advanced breast, prostate, and other cancer sites. The data were extracted, and point estimates for the primary and secondary trial endpoints were converted into the NNT parameter.ResultsNNT represents the number of patients that need to be treated with a new intervention in order to avoid one additional patient developing the event and is a powerful approach that can be used to make sense of numerical results from clinical trials. In patients with advanced breast, prostate, and other cancer sites, 18, 22, and 21 patients, respectively, would need to be treated with denosumab for at least 24xa0months to avoid one patient developing an SRE.ConclusionsThe NNT approach is a simple and effective method to express the findings of randomized trials in a clinically meaningful way. In this analysis, the incremental benefits of denosumab would be realized when a minimum of 18 to 22 patients are treated for a prolonged duration. Clinicians would have to weigh the costs and benefits between denosumab and zoledronic acid when bone-targeted therapy is indicated.

Absence of methylation-dependent transcriptional silencing in TP73 irrespective of the methylation status of the CDKN2A CpG island in plasma cell neoplasia

Few studies exist regarding the methylation status of the TP73 CpG island in plasma cell dyscrasias. We have tested whether CpG methylation of both CDKN2A and TP73 occurs in 45 individuals with multiple myeloma (24 male and 21 female, mean age 66.4 years) and in 4 patients (2 male and 2 female, mean age 61.7 years) with Waldenströms macroglobulinemia. No patient was found to be methylated for the promoter of TP73 while CDKN2A promoter was found to be methylated in 12/45 MM patients (26.6%) at diagnosis and in 1/4 WM patients. To verify the absence of detectable methylation observed using MSP, we performed bisulphite sequence analysis on a subset of the cases and confirmed the absence of methylation. Interesting trends were identified where patients with methylated CDKN2A had an increased risk of death (HR = 1.9, p = 0.32), advanced stage disease (DS > or = II) (OR = 1.9, p = 0.3) and anemia (OR = 1.4, p = 0.6). TP73 CpG methylation is an infrequent event in patients with MM and WM. Further evaluation in a larger sample of patients is needed in order to enhance our statistical power and to test our hypothesis that CDKN2A methylation status can become a useful prognostic biomarker.

Bcl2-interacting killer CpG methylation in multiple myeloma: a potential predictor of relapsed/refractory disease with therapeutic implications

Abstract BIK (bcl2-interacting killer) is the founding member of the BH3-only bcl-2 family of pro-apoptotic proteins, which is suppressed in various cancers. In multiple myeloma (MM), BIK has been shown to be epigenetically silenced in vitro, but there is a lack of clinical data. We investigated the CpG methylation status of the BIK promoter in a well-characterized clinical series of patients with MM and investigated its clinical relevance. Forty patients with MM (21 male, 19 female; mean age 66) were studied. According to the International Staging System (ISS) they were classified as 16 patients with stage I, 12 patients with stage II and 12 patients with stage III disease. Methylation in the BIK CpG island was assessed by methylation-specific polymerase chain reaction (MSP) assay. Logistic regression analysis was used to investigate associations between gene methylation and age, ISS stage, performance status, extramedullary disease, bone disease, anemia (hemoglobin ≤10 mg/dL), serum albumin, β2-microglobulin level and relapsed/refractory disease. Methylation in the BIK CpG island was detected in 16 patients (40%), with a trend favoring male gender (odds ratio [OR] =u20093.08, p =u20090.09) and development of bone disease and extramedullary disease (OR =u20091.6, p =u20090.35 and OR =u20093, p =u20090.14, respectively). Patients with MM with methylated BIK CpG island had a statistically significant risk for disease evolution to relapsed/refractory disease (OR =u20095.4, p =u20090.03). This study provides clinical evidence that methylation-induced transcriptional silencing of the BIK pro-apoptotic gene may occur in MM, which might serve as a predictor of the development of relapsed/refractory MM. These findings warrant validation in larger cohorts of patients and suggest therapeutic utility for agents that enhance BIK expression.

Improving the Cost Efficiency of PD-1/PD-L1 Inhibitors for Advanced Urothelial Carcinoma: A Major Role for Precision Medicine?

Five Programmed Death (PD)-1 and PD-ligand (L)-1 inhibitors are now approved by the US Food and Drug Administration for treating patients with advanced urothelial carcinoma (UC) following platinum-based chemotherapy [1–5]. Pembrolizumab is fully approved based on an extension of overall survival (OS) in a phase III trial, while the other four agents (atezolizumab, durvalumab, nivolumab, and avelumab) have gained accelerated approval based on the objective response rate (ORR) in 15–20% of patients, most of which are exceedingly durable. Indeed, in a recent update of the Keynote (KN)-045 phase III trial after an extended median follow-up of 27.7 mo, pembrolizumab yielded a more robust extension of median OS (10.3 vs 7.3 mo; hazard ratio [HR], 0.70; p < 0.0002), better ORR (21.1% vs 11.0%), and median duration of response (not reached [range 1.6+ to 30.3+ mo] vs 4.4 mo [1.4+ to 29.9+ mo]) compared with chemotherapy [6]. However, the overall progression-free survival (PFS) was not different (2.1 vs 3.3 mo; HR, 0.96; p = 0.32). The toxicity profile was better for pembrolizumab when examining toxicities of any grade (62.0% vs 90.6%) and grade 3 adverse events (16.5% vs 50.2%). In this months issue of European Urology, Sarfaty and colleagues [7] developed a Markov model to compare the cost efficacy of pembrolizumab versus chemotherapy for patients enrolled in the KN-045 trial. Drug costs were acquired for the USA, the UK, Canada, and Australia, and converted to US dollars at the exchange rates in September 2017. Pembrolizumab conferred a gain of 0.36–0.37 in quality-adjusted life-years (QALYs) compared with