PanDrugs2: prioritizing cancer therapies using integrated individual multi-omics data.

Genomics studies routinely confront researchers with long lists of tumor alterations detected in patients. Such lists are difficult to interpret since only a minority of the alterations are relevant biomarkers for diagnosis and for designing therapeutic strategies. PanDrugs is a methodology that facilitates the interpretation of tumor molecular alterations and guides the selection of personalized treatments. To do so, PanDrugs scores gene actionability and drug feasibility to provide a prioritized evidence-based list of drugs. Here, we introduce PanDrugs2, a major upgrade of PanDrugs that, in addition to somatic variant analysis, supports a new integrated multi-omics analysis which simultaneously combines somatic and germline variants, copy number variation and gene expression data. Moreover, PanDrugs2 now considers cancer genetic dependencies to extend tumor vulnerabilities providing therapeutic options for untargetable genes. Importantly, a novel intuitive report to support clinical decision-making is generated. PanDrugs database has been updated, integrating 23 primary sources that support >74K drug-gene associations obtained from 4642 genes and 14 659 unique compounds. The database has also been reimplemented to allow semi-automatic updates to facilitate maintenance and release of future versions. PanDrugs2 does not require login and is freely available at https://www.pandrugs.org/.

RANK is a poor prognosis marker and a therapeutic target in ER-negative postmenopausal breast cancer.

Despite strong preclinical data, the therapeutic benefit of the RANKL inhibitor, denosumab, in breast cancer patients, beyond the bone, is unclear. Aiming to select patients who may benefit from denosumab, we hereby analyzed RANK and RANKL protein expression in more than 2,000 breast tumors (777 estrogen receptor-negative, ER- ) from four independent cohorts. RANK protein expression was more frequent in ER- tumors, where it associated with poor outcome and poor response to chemotherapy. In ER- breast cancer patient-derived orthoxenografts (PDXs), RANKL inhibition reduced tumor cell proliferation and stemness, regulated tumor immunity and metabolism, and improved response to chemotherapy. Intriguingly, tumor RANK protein expression associated with poor prognosis in postmenopausal breast cancer patients, activation of NFKB signaling, and modulation of immune and metabolic pathways, suggesting that RANK signaling increases after menopause. Our results demonstrate that RANK protein expression is an independent biomarker of poor prognosis in postmenopausal and ER- breast cancer patients and support the therapeutic benefit of RANK pathway inhibitors, such as denosumab, in breast cancer patients with RANK+ ER- tumors after menopause.

CALR-mutated cells are vulnerable to combined inhibition of the proteasome and the endoplasmic reticulum stress response.

Cancer is driven by somatic mutations that provide a fitness advantage. While targeted therapies often focus on the mutated gene or its direct downstream effectors, imbalances brought on by cell-state alterations may also confer unique vulnerabilities. In myeloproliferative neoplasms (MPN), somatic mutations in the calreticulin (CALR) gene are disease-initiating through aberrant binding of mutant CALR to the thrombopoietin receptor MPL and ligand-independent activation of JAK-STAT signaling. Despite these mechanistic insights into the pathogenesis of CALR-mutant MPN, there are currently no mutant CALR-selective therapies available. Here, we identified differential upregulation of unfolded proteins, the proteasome and the ER stress response in CALR-mutant hematopoietic stem cells (HSCs) and megakaryocyte progenitors. We further found that combined pharmacological inhibition of the proteasome and IRE1-XBP1 axis of the ER stress response preferentially targets Calr-mutated HSCs and megakaryocytic-lineage cells over wild-type cells in vivo, resulting in an amelioration of the MPN phenotype. In serial transplantation assays following combined proteasome/IRE1 inhibition for six weeks, we did not find preferential depletion of Calr-mutant long-term HSCs. Together, these findings leverage altered proteostasis in Calr-mutant MPN to identify combinatorial dependencies that may be targeted for therapeutic benefit and suggest that eradicating disease-propagating Calr-mutant LT-HSCs may require more sustained treatment.

Subfoveal choroidal thickness as a potential predictor of treatment response after intravitreal ranibizumab injections for polypoidal choroidal vasculopathy.

OBJECTIVE: To evaluate the impact of subfoveal choroidal thickness (SFCT) and other clinical biomarkers in intravitreal anti-vascular endothelial growth factor response in treatment-naive Caucasian patients diagnosed with polypoidal choroidal vasculopathy (PCV/AT1). DESIGN: Cross-sectional study. PARTICIPANTS: Treatment-naive patients diagnosed with PCV/AT1 recruited in a single centre from January 2013 to December 2020. METHODS: Eligibility was determined in treatment-naive PCV patients who received a loading dose of 3 injections of 0.5 mg ranibizumab. A diagnosis of PCV/AT1 was made based on the diagnostic criteria in the efficacy and safety of verteporfin photodynamic therapy in combination with ranibizumab or alone versus ranibizumab monotherapy in patients with sumptomatic macular polypoidal choroidal vasculopathy study. Choroidal thickness was manually measured by enhanced depth imaging technology in Spectralis spectral domain optical coherence tomography. RESULTS: Eighty-three eyes of 83 patients were included in this study, 47 patients diagnosed with PCV/AT1 with a good response to 3 intravitreal injections of ranibizumab and 36 with a poor response. The receiver operating characteristic curve of treatment effect against the SFCT revealed that the area under the curve was 0.85 (range, 0.74-0.96). Based on the Youden index, the optimal SFCT cut-off point for predicting a poor response to anti-vascular endothelial growth factor is 257 µm. In the multivariate analysis, the SFCT remained statistically significant (odds ratio 1.02 [range, 1.01-1.04]; P = 0.008). The combined effect of treatment effect against clinical biomarkers produced an area under the curve of 0.90 (range, 0.82-0.98). CONCLUSION: SFCT is a risk factor for a poor response to the 3 loading injections of ranibizumab in treatment-naive PCV/AT1 Caucasian patients. A cut-off point of 257 µm could be a valuable parameter for defining the population at risk for an inadequate response to ranibizumab.

Spontaneous resolution of inflammatory epiretinal membrane: Case series and review of the literature.

Epiretinal membrane (ERM) represents a common complication of uveitis that may contribute independently to vision loss in patients with uveitis. Although spontaneous idiopathic ERM separation has been previously reported, to the best of our knowledge there are only two case reports in the scientific literature that depicts spontaneous regression of an inflammation-associated ERM. Spontaneous ERM separation is a rare but possible event, which occurs most often subsequent to posterior vitreous detachment. We present a case series of three patients with uveitis that exhibit the formation and subsequent spontaneous resolution of an inflammatory ERM.

Bioinformatics roadmap for therapy selection in cancer genomics.

Tumour heterogeneity is one of the main characteristics of cancer and can be categorised into inter- or intratumour heterogeneity. This heterogeneity has been revealed as one of the key causes of treatment failure and relapse. Precision oncology is an emerging field that seeks to design tailored treatments for each cancer patient according to epidemiological, clinical and omics data. This discipline relies on bioinformatics tools designed to compute scores to prioritise available drugs, with the aim of helping clinicians in treatment selection. In this review, we describe the current approaches for therapy selection depending on which type of tumour heterogeneity is being targeted and the available next-generation sequencing data. We cover intertumour heterogeneity studies and individual treatment selection using genomics variants, expression data or multi-omics strategies. We also describe intratumour dissection through clonal inference and single-cell transcriptomics, in each case providing bioinformatics tools for tailored treatment selection. Finally, we discuss how these therapy selection workflows could be integrated into the clinical practice.

Whole-genome CRISPR screening identifies N-glycosylation as a genetic and therapeutic vulnerability in CALR-mutant MPNs.

Calreticulin (CALR) mutations are frequent, disease-initiating events in myeloproliferative neoplasms (MPNs). Although the biological mechanism by which CALR mutations cause MPNs has been elucidated, there currently are no clonally selective therapies for CALR-mutant MPNs. To identify unique genetic dependencies in CALR-mutant MPNs, we performed a whole-genome clustered regularly interspaced short palindromic repeats (CRISPR) knockout depletion screen in mutant CALR-transformed hematopoietic cells. We found that genes in the N-glycosylation pathway (among others) were differentially depleted in mutant CALR-transformed cells as compared with control cells. Using a focused pharmacological in vitro screen targeting unique vulnerabilities uncovered in the CRISPR screen, we found that chemical inhibition of N-glycosylation impaired the growth of mutant CALR-transformed cells, through a reduction in MPL cell surface expression. We treated Calr-mutant knockin mice with the N-glycosylation inhibitor 2-deoxy-glucose (2-DG) and found a preferential sensitivity of Calr-mutant cells to 2-DG as compared with wild-type cells and normalization of key MPNs disease features. To validate our findings in primary human cells, we performed megakaryocyte colony-forming unit (CFU-MK) assays. We found that N-glycosylation inhibition significantly reduced CFU-MK formation in patient-derived CALR-mutant bone marrow as compared with bone marrow derived from healthy donors. In aggregate, our findings advance the development of clonally selective treatments for CALR-mutant MPNs.

Impact of ocular dominance on circumpapillary and macular retinal nerve fibre layer thickness and ganglion cell layer thickness in a healthy pediatric population.

OBJECTIVE: This study was designed to evaluate potential differences in circumpapillary retinal nerve fibre layer (cpRNFL) thickness and segmented macular retinal layers between dominant and nondominant eyes on spectral-domain optical coherence tomography in a pediatric population. DESIGN: Cross-sectional study. PARTICIPANTS: 89 healthy children attending a general pediatric clinic. METHODS: Participants underwent sighting dominant testing and macular and cpRNFL spectral-domain optical coherence tomography. Segmented macular layer thicknesses and cpRNFL thickness were compared for individual patients based on their ocular dominance. RESULTS: Ocular dominance occurred particularly in the right eye (64.7%). Dominant and nondominant eyes did not differ significantly in axial length or spherical equivalent refraction; axial length: 22.99 ± 1.17 mm versus 22.98 ± 1.19 mm; p = 0.51 and spherical equivalent refraction: -0.09 ± 2.68 D versus 0.32 ± 2.93 D; p = 0.41. In the comparison of the macular ganglion layer the average thickness in the 1 mm central Early Treatment Diabetic Retinopathy Study area was significantly different between the dominant and nondominant eye (16.56 ± 6.02 µm vs 17.58 ± 8.32 µm; p = 0.02). However, when compensating with Bonferroni, this difference was no longer statistically significant. There were no differences in the analyses of average global and sectorial cpRNFL thickness in dominant and nondominant eyes. CONCLUSION: Dominant eyes demonstrated no significantly thicker average macular retinal nerve fiber layer (mRNFL), Ganglion cell layer (GCL) thickness or cpRNFL thickness. No ocular characteristic was found to be associated with the relative dominance of an eye in eyes with low anisometropia.

bollito: a flexible pipeline for comprehensive single-cell RNA-seq analyses.

SUMMARY: bollito is an automated, flexible and parallelizable computational pipeline for the comprehensive analysis of single-cell RNA-seq data. Starting from FASTQ files or preprocessed expression matrices, bollito performs both basic and advanced tasks in single-cell analysis integrating >30 state-of-the-art tools. This includes quality control, read alignment, dimensionality reduction, clustering, cell-marker detection, differential expression, functional analysis, trajectory inference and RNA velocity. bollito is built using the Snakemake workflow management system, which easily connects each execution step and facilitates the reproducibility of results. bollito's modular design makes it easy to incorporate other packages into the pipeline enabling its expansion with new functionalities. AVAILABILITY AND IMPLEMENTATION: Source code is freely available at https://gitlab.com/bu_cnio/bollito under the MIT license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Beyondcell: targeting cancer therapeutic heterogeneity in single-cell RNA-seq data.

We present Beyondcell, a computational methodology for identifying tumour cell subpopulations with distinct drug responses in single-cell RNA-seq data and proposing cancer-specific treatments. Our method calculates an enrichment score in a collection of drug signatures, delineating therapeutic clusters (TCs) within cellular populations. Additionally, Beyondcell determines the therapeutic differences among cell populations and generates a prioritised sensitivity-based ranking in order to guide drug selection. We performed Beyondcell analysis in five single-cell datasets and demonstrated that TCs can be exploited to target malignant cells both in cancer cell lines and tumour patients. Beyondcell is available at: https://gitlab.com/bu_cnio/beyondcell .

Delayed Onset of Retinal Embolism After Coronary Angioplasty.

Although rare, retinal artery cholesterol emboli (Hollenhorst plaques) post angioplasty can be a severe event that can cause irreversible visual disturbances, usually taking place during or following the procedure. However, as the number of procedures continues to increase, interventional radiologists or cardiologists who perform coronary catheterization should be cognizant of its possible delayed occurrence, as in the case presented herein.

Synergic effect of corneal hysteresis and central corneal thickness in the risk of early-stage primary open-angle glaucoma progression.

PURPOSE: To evaluate corneal hysteresis (CH), acquired with ocular response analyzer (ORA), as a risk factor for glaucoma progression in early-stage primary open-angle glaucoma (POAG). METHODS: In a historical cohort study, patients diagnosed in 2011 with early-stage POAG according to the Hodapp, Parrish and Anderson classification modified for Octopus perimetry and followed up until glaucomatous progression development; otherwise, observations were censored in October 2018. Cox regression was used to obtain hazard ratios (HR) to evaluate baseline variables (CH, central corneal thickness, gender, age IOP and glaucoma family history) as risk factors for perimetric glaucoma progression. A likelihood ratio test for interaction was performed in order to assess the effect of the combination of CH and CCT on the risk of progression. RESULTS: Of the cohort of 1573 patients, 11.38% developed early-stage POAG progression during the follow-up. The mean follow-up time was 3.28 ± 1.92 years. Patients without progression had a higher CH (11.35 ± 1.43 vs 9.07 ± 1.69 mmHg; p < 0.001) and CCT (570.75 ± 17.71 vs 554.51 ± 23.20; p < 0.001). In the multivariate analysis, each 1 mmHg of lower CH was associated with an increase of 2.13 times in the HR of progression (95% CI: 1.92-2.32; p < 0.001). CH hazard ratio was modified by CCT, with higher values of CCT and CH resulting in a higher HR of early glaucoma progression (p < 0.001). CONCLUSIONS: CH can be considered as a risk factor of progression in early-stage POAG. The risk associated with CH changed depending on CCT values, acting synergistically slowing the risk of glaucoma progression with higher values.

Reproducibility of macular retinal nerve fiber layer and ganglion cell layer thickness measurements in a healthy pediatric population.

PURPOSE: To evaluate the reproducibility of Spectralis spectral domain-OCT segmented ganglion cell layer (GCL) and macular retinal nerve fiber layer (mRNFL) measurements in a healthy children population. METHODS: An observational, cross-sectional study was carried out on 79 children to measure the intraobserver reproducibility and the repeatability between a novel and an experienced operator of the segmented macular GCL and mRNFL. Interobserver and intraobserver reproducibility were defined by the intraclass correlation coefficient (ICC) and coefficients of variation (COV). Kruskal-Wallis test was used to determine statistical significance in the COV of three age groups children (younger than 6 years, between 6 and 12, and older than 12 years old). RESULTS: The results from the intraoperator GCL thickness analysis were highly reproducible (COV < 6%) and reliable (ICCs > 0.81). When the measurements were compared between a novel and an experienced examiner lower ICCs and higher COV were found. COVs ranged from 1.85% (total volume area) to 5.57% at the central ETDRS subfield while the ICC vary from 0.632 (outer inferior) to 0.832 (inner inferior). The repeatability and reproducibility of the mRNFL thickness were lower, with ICCs ranging from 0.428 to 0.872 in the interobserver analysis and from 0.897 to 0.346 in the interobserver one. CONCLUSION: In the present study, we establish substantial reliability of the GCL thickness in children with Spectralis® SD-OCT in all the sectors, albeit lower than the reported in the literature with other SD-OCTs and in adults. The reproducibility and repeatability of the mRNFL were significantly lower. We were unable to find consistent statistical significant differences between the COV of the three age groups.

Atypical perifoveal exudative vascular anomalous complex (PEVAC) with multifocal and bilateral presentation.

PURPOSE: To report an unusual case of multifocal and bilateral perifoveal exudative vascular anomalous complex (PEVAC) lesions in an otherwise healthy patient. OBSERVATIONS: A 62-year-old male with complaints of long-term progressive blurred vision in both eyes. There was no history of diabetes, arterial hypertension or inflammatory disease. Best-corrected visual acuity was 75 and 80 letters in his right eye (OD) and left eye (OS) respectively. Fundus examination showed three perifoveal isolated large aneurysmal lesions in his OD and an additional one in his OS, associated with smaller microaneurysms, haemorrhages and intraretinal exudation. On optical coherence tomography PEVAC appeared as a round lesions with a hyperreflective wall and lumen containing variably reflective material, typically surrounded by intraretinal fluid. Dye-based angiography demonstrated three and one well-defined large hyperfluorescent aneurysmal lesions in his OD and OS respectively, with no leakage. The optical coherence tomography angiography images showed flow signal within the intraretinal aneurysmal lesions. No anomalous flow signal in the outer retina and choriocapillaris was present. After one bilateral anti-vascular endothelial growth factor intravitreal injection no anatomical or functional improvement was observed, and no changes in the lesions or visual acuity were observed at 6 months follow-up. CONCLUSIONS AND IMPORTANCE: We describe an atypical case of PEVAC, an uncommon and poorly known macular disorder, with bilateral presentation and multifocal lesions in one eye. To our knowledge, this is the first reported case of bilateral PEVAC lesions. Hence, PEVAC should be included in the differential diagnosis of bilateral perifoveal aneurysmal lesions with macular edema.

In Silico Drug Prescription for Targeting Cancer Patient Heterogeneity and Prediction of Clinical Outcome.

In silico drug prescription tools for precision cancer medicine can match molecular alterations with tailored candidate treatments. These methodologies require large and well-annotated datasets to systematically evaluate their performance, but this is currently constrained by the lack of complete patient clinicopathological data. Moreover, in silico drug prescription performance could be improved by integrating additional tumour information layers like intra-tumour heterogeneity (ITH) which has been related to drug response and tumour progression. PanDrugs is an in silico drug prescription method which prioritizes anticancer drugs combining both biological and clinical evidence. We have systematically evaluated PanDrugs in the Genomic Data Commons repository (GDC). Our results showed that PanDrugs is able to establish an a priori stratification of cancer patients treated with Epidermal Growth Factor Receptor (EGFR) inhibitors. Patients labelled as responders according to PanDrugs predictions showed a significantly increased overall survival (OS) compared to non-responders. PanDrugs was also able to suggest alternative tailored treatments for non-responder patients. Additionally, PanDrugs usefulness was assessed considering spatial and temporal ITH in cancer patients and showed that ITH can be approached therapeutically proposing drugs or combinations potentially capable of targeting the clonal diversity. In summary, this study is a proof of concept where PanDrugs predictions have been correlated to OS and can be useful to manage ITH in patients while increasing therapeutic options and demonstrating its clinical utility.

Influence of mutation bias and hydrophobicity on the substitution rates and sequence entropies of protein evolution.

The number of amino acids that occupy a given protein site during evolution reflects the selective constraints operating on the site. This evolutionary variability is strongly influenced by the structural properties of the site in the native structure, and it is quantified either through sequence entropy or through substitution rates. However, while the sequence entropy only depends on the equilibrium frequencies of the amino acids, the substitution rate also depends on the exchangeability matrix that describes mutations in the mathematical model of the substitution process. Here we apply two variants of a mathematical model of protein evolution with selection for protein stability, both against unfolding and against misfolding. Exploiting the approximation of independent sites, these models allow computing site-specific substitution processes that satisfy global constraints on folding stability. We find that site-specific substitution rates do not depend only on the selective constraints acting on the site, quantified through its sequence entropy. In fact, polar sites evolve faster than hydrophobic sites even for equal sequence entropy, as a consequence of the fact that polar amino acids are characterized by higher mutational exchangeability than hydrophobic ones. Accordingly, the model predicts that more polar proteins tend to evolve faster. Nevertheless, these results change if we compare proteins that evolve under different mutation biases, such as orthologous proteins in different bacterial genomes. In this case, the substitution rates are faster in genomes that evolve under mutational bias that favor hydrophobic amino acids by preferentially incorporating the nucleotide Thymine that is more frequent in hydrophobic codons. This appearingly contradictory result arises because buried sites occupied by hydrophobic amino acids are characterized by larger selective factors that largely amplify the substitution rate between hydrophobic amino acids, while the selective factors of exposed sites have a weaker effect. Thus, changes in the mutational bias produce deep effects on the biophysical properties of the protein (hydrophobicity) and on its evolutionary properties (sequence entropy and substitution rate) at the same time. The program Prot_evol that implements the two site-specific substitution processes is freely available at https://ub.cbm.uam.es/prot_fold_evol/prot_fold_evol_soft_main.php#Prot_Evol.

Diagnostic capacity of SD-OCT segmented ganglion cell complex versus retinal nerve fiber layer analysis for congenital glaucoma.

PURPOSE: To compare the diagnostic performance of circumpapillary retinal nerve fiber layer (cpRNFL) analysis versus segmented ganglion cell complex analysis both by spectral-domain optical coherence tomography (SD-OCT) in children with primary congenital glaucoma (PCG). METHODS: Participants were 40 children diagnosed with PCG and 60 healthy children. Ophthalmological data collected (for one eye per child) were cup-disc ratio (C/D) and axial length (AL). SD-OCT with automated segmentation was used to measure the thicknesses and volumes of the macular retinal nerve fiber layer (mRNFL), ganglion cell layer (GCL), and inner plexiform layer (IPL). For the cpRNFL measurements conventional S-D OCT software was used and the capacity of each method to discriminate between normal and glaucomatous eyes was compared. RESULTS: Mean age was 11.20 ± 3.94 years for the glaucoma patients and 10.90 ± 2.46 years for controls (p = 0.64). All measurements were reduced (thinner) in the glaucoma group, significantly so for: cpRNFL, GCL, IPL and outer-superior and outer-inferior quadrant mRNFL. According to their areas under the receiver operating characteristics curve (AUC), temporal superior cpRNFL (0.869) and outer superior GCL (0.840), IPL (0.799), and mRNFL (0.767) showed the better diagnostic capacity. No differences were observed in AUCs for the most discriminatory cpRNFL and macular measurements. CONCLUSION: Segmented macular layer analysis shows a good capacity to discriminate between normal and glaucomatous eyes; which is comparable to that of cpRNFL analysis in children with PCG.

Interferon-associated retinopathy in a patient with metastatic melanoma.

We present the unusual case of a 35 year-old woman with stage IV melanoma and widespread metastases, who was undergoing treatment with interferon alpha-2b and who presented with interferon-associated retinopathy. The patient, who had been taking interferon treatment for three months, complained of a sudden loss of visual acuity in the left eye. An ocular examination revealed multiple cotton wool spots along the retina and macular involvement. Interferon treatment was suspended. Although rare, retinopathy represents a potentially serious adverse effect of interferon treatment. Although normally patients are asymptomatic, complications derived of its use may arise, which can lead to significant visual impairment. We therefore suggest that before initiating treatment with this drug, patients should be informed of its potential ocular risks, and that regular eye examinations are conducted along with the treatment.

Interferon-associated retinopathy in a patient with metastatic melanoma / Retinopatia relacionada ao interferon em paciente com melanoma metastático

We present the unusual case of a 35 year-old woman with stage IV melanoma and widespread metastases, who was undergoing treatment with interferon alpha-2b and who presented with interferon-associated retinopathy. The patient, who had been taking interferon treatment for three months, complained of a sudden loss of visual acuity in the left eye. An ocular examination revealed multiple cotton wool spots along the retina and macular involvement. Interferon treatment was suspended. Although rare, retinopathy represents a potentially serious adverse effect of interferon treatment. Although normally patients are asymptomatic, complications derived of its use may arise, which can lead to significant visual impairment. We therefore suggest that before initiating treatment with this drug, patients should be informed of its potential ocular risks, and that regular eye examinations are conducted along with the treatment.

Apresentamos o caso de uma mulher de 35 anos com melanoma em estágio IV e metástases generalizadas tratados com interferon alpha-2b, que proporcionou uma retinopatia associada ao interferon. Mulher de 35 anos de idade tratados com interferon durante os últimos três meses apresentou uma perda súbita da acuidade visual no olho esquerdo. Exame ocular revelou vários pontos de algodão ao longo da retina e mácula. Tratamento com interferon foi parado. Retinopatia associada ao uso de interferon está entre os possíveis efeitos colaterais, embora rara, não deve ser subestimada. Embora geralmente assintomática, complicações decorrentes de seu uso podem levar à perda visual significativa. Consideramos, portanto, que antes de iniciar o tratamento com este medicamento, os pacientes devem ser informados sobre os riscos potenciais e os exames oftalmológicos são recomendados durante cada tratamento.