Ana Milovanovic

The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts.

ABSTRACT: RNA homeostasis is dysregulated in cancer and affects disease progression and therapy resistance. N6-methyladenosine (m6A), the most abundant epitranscriptomic modification in eukaryotic messenger RNA, plays a pivotal role in RNA biology, affecting transcript stability, translation, and splicing. Our study uncovers the extensive m6A changes in patients with T-cell acute lymphoblastic leukemia (T-ALL), to our knowledge, for the first time. It reveals m6A's regulatory role in the oncogenic MYC and cholesterol biosynthesis pathways. In addition, we discovered that T-ALL is highly dependent on the m6A reader heterogeneous nuclear ribonucleoprotein C (HNRNPC). HNRNPC is transcriptionally controlled by MYC and is an essential regulator of m6A-modified transcripts. Consequently, transcriptional silencing of HNRNPC profoundly impairs oncogenic pathways and critically diminishes leukemia cell growth. In addition, the levels of the m6A demethylase fat mass and obesity-associated protein (FTO) are significantly elevated in T-ALL cells compared with normal cells, and to other types of leukemia. Targeting FTO shows therapeutic potential in preclinical disease models and synergizes with clinically relevant therapeutics. Our findings underscore the integral role of RNA methylation in orchestrating cancer cell oncogene expression and metabolism and highlight promising novel therapeutic avenues for the treatment of T-cell leukemia.

ABSTRACT Nanopore direct RNA sequencing (DRS) holds promise for advancing our understanding of the epitranscriptome by detecting RNA modifications in native RNA molecules. Recently, Oxford Nanopore Technologies (ONT) has released basecalling models capable of detecting several RNA modifications. However, their accuracy, sensitivity and specificity, as well as cross-reactivity against other modification types, remains largely unexplored. Here, we systematically benchmark modification-aware models by evaluating their performance on a highly-multiplexed panel of synthetic molecules covering all possible sequence contexts, as well as on biological samples from a diverse set of species. We find that modification-aware models reliably detect diverse RNA modification types across a broad range of sequence contexts. However, they are prone to elevated false positive rates and exhibit notable cross-reactivity with other RNA modification types. We show that the use of modification-free controls allows significant, yet incomplete removal of false positives, thus constituting an essential control. Finally, we demonstrate that basecalling error patterns and alterations in current features can identify differentially modified sites, for modifications for which modification-aware models are absent. Overall, our results underscore the utility and accuracy of modification-aware basecalling models for RNA modification detection, while highlighting the importance of including diverse control samples to mitigate false positive rates.

Introduction: Preliminary evidence suggests that neprilysin inhibitors (ARNI) may affect angiogenesis in heart failure with reduced ejection fraction (HFrEF). Hypothesis: Since CD34+ cells represent one of the key determinants of vasculogenesis and angiogenesis, we investigated a potential association between clinical effects of ARNI and circulating CD34+ cell count in HFrEF. Methods: We performed a prospective single center pilot cohort study in 27 HFrEF patients. After enrollment, all patients received standard heart failure therapy including ACE inhibitors (ACE-I) or angiotensin receptor blockers (ARB) for 3 months. At this point, ACE-I/ARB were switched to ARNI. At the time of switch, we measured circulating CD34+ cell count using Beckman-Coulter Navios EX flow cytometry with standard antibodies according to ISAGE protocol. All patients were followed for 6 months after the switch. Favorable response to ARNI therapy was defined as an increase in left ventricular ejection fraction (LVEF) ≥5% at 6-month follow-up. Results: At 6 months we found a favorable response to ARNI in 18 patients (64%, Group A), and 10 had less favorable response (36%, Group B). Before ARNI introduction the groups did not differ in gender (male: 94% in Group A vs. 78% in Group B, P=0.21), heart failure etiology (ischemic: 35% vs. 23%, P=0.49), diabetes (11% vs. 22%, P=0.46), renal dysfunction (22% vs. 43%, P=0.25), hypertension (28% vs. 55%, P=0.17), LVEF (36±5% vs. 34±7%, P=0.41), LVEDV (181±57 mL vs. 185±36 mL, P=0.83), TAPSE (1.8±0.8 vs. 1.9±0.4, P=0.91) or NT-proBNP (980±1438 pg/mL vs. 780±603 pg/mL, P=0.70). Compared to Group B, patients in Group A were younger (52±13 years vs. 64±9 years, P=0.03) and had a higher CD34+ cell count (2.9±2.0x106/L vs. 1.2±0.5x106/L, P=0.02). When compared to the remainig cohort, patients with CD34+ cell count above the median displayed a significantly higher increase in LVEF (+8.1±3.2% vs. +4.7±3.7%, P=0.03), and a trend of increase in TAPSE (+0.4±0.4 cm vs. -0.1±0.7 cm, P=0.06). Conclusion: Higher numbers of circulating CD34+ cells appear to be associated with more pronounced beneficial effects of ARNI therapy in HFrEF patients. Thus, the effects or ARNI in heart failure may be partly mediated through modulation of vascular homeostasis.

Background and aims: Pro-inflammatory cytokines may play a role in the pathogenesis of CD. The differential production of cytokines has been related to genetic polymorphisms in gene promoter regions. The aim of this case-control study was to assess the relationship between polymorphisms in cytokines genes and CD susceptibility as well as disease phenotype. Methods: 78 patients with CD and 102 controls were included in the study. 49 CD patients had inactive disease according to the Harvey-Bradshaw index (HBI) whereas the remaining 29 had an index 4-7.Cytokine genotyping was performed using PCR and PCR/RFLP. Gene polymorphisms that were probed for included IL-1beta-511C/T, IL-1beta+3953C/T, IL-1RaVNTR, TNFal-pha857C/T, TNFalpha308G/A, LTalpha+252A/G and IL-6-174G/C. Results: A significant association was observed between higher HBI and perforating disease (p=0.01) as well as disease onset >40 years (p=0.004). As compared to controls, we observed that carriers of the homozygous variant for TNFalpha857TT and for IL-6-174 CC had an increased risk for CD: OR 4.14 (95% IC 0.81-21.24) and OR 10.39 (95% IC 2.22-48.77), respectively. In respect to phenotype, significant associations were observed between TNFalpha857 polymorphism and the existence of perianal disease (p=0.002), recto-vaginal fistula (0.04), the presence of metabolic bone disease (p=0.05), as well as with age of disease onset (p=0.07). The latter was significantly associated to LTalpha +252 A/G and IL-6-174 G/C polymorphisms as well (p=0.05 and 0.04, respectively). Gender distribution in CD patients was associated to TNFal-pha308 polymorphism (p=0.004) whereas disease activity was significantly related to polymorphism in the sole anti-inflammatory cytokine analysed -IL-1RaVNTR (p=0.02).