ObjectivesUlcerative colitis (UC) is a chronic inflammatory disorder of unknown aetiology. Gut vi... more ObjectivesUlcerative colitis (UC) is a chronic inflammatory disorder of unknown aetiology. Gut virome dysbiosis is fundamental in UC progression, although its role in the early phases of the disease is far from fully understood. Therefore, we sought to investigate the role of a virome-associated protein encoded by theOrthohepadnavirusgenus, the hepatitis B virus X protein (HBx), in UC aetiopathogenesis.DesignHBx positivity of UC patient-derived blood and gut mucosa was assessed by RT-PCR and Sanger sequencing and correlated with clinical characteristics by multivariate analysis. Transcriptomics was performed on HBx-overexpressing endoscopic biopsies from healthy donors.C57BL/6 mice underwent intramucosal injections of liposome-conjugated HBx-encoding plasmids or the control, with or without antibiotic treatment. Multidimensional flow cytometry analysis was performed on colonic samples from HBx-treated and control animals. Transepithelial electrical resistance measurement, proliferat...
Resistance to imatinib represents an important scientific and clinical issue in CML and Ph+ ALL a... more Resistance to imatinib represents an important scientific and clinical issue in CML and Ph+ ALL and is usually due to the selection of cells harbouring kinase point mutations of the BCR-ABL gene or presenting BCR-ABL gene amplification. In order to improve Bcr-Abl inhibition, several inhibitors with higher potency compared to imatinib have been identified including dasatinib and nilotinib, which show clinical activity in patients resistant to imatinib. In the present study the effects of the novel inhibitor SKI-606 on various models of resistance to imatinib were studied. SKI-606 is a selective dual Src/Abl inhibitor potently active against several CML cell lines and transfectants with IC50 values in the low nanomolar range, 2 logs lower than those obtained with imatinib. Cells expressing activated forms of c-KIT (HMC1560 and GIST882) or PDGFR (Ba/F3 Tel-PDGFRβ) were unaffected by SKI-606, in contrast to imatinib and dasatinib. SKI-606 reduced proliferation rate of cells where resistance to imatinib was caused by BCR-ABL gene amplification, such as Lama84R and of imatinib-resistant K562R and KCL22R in which no known mechanism have been identified. D276G, Y253F and E255K kinase point mutants were affected by SKI-606, resulting in inhibition of cell proliferation with IC50 values of 25, 40 and 394 nM (imatinib:1147, 1888 and 3174 nM). No activity was observed against T315I mutant. These results were confirmed in in vivo experiments, in models where resistance was not caused by mutations, as well as in cells carrying the Y253F, E255K and D276G mutations. In the first one SKI-606 was able to eradicate tumors, while significant delay in tumor growth was observed using Ba/F3 transfectants. Modelling considerations attribute the superior activity of SKI-606 to its ability to bind the intermediate (1M52) conformation of Bcr-Abl, in contrast to imatinib. Excluding the T315 residue involved in direct hydrophilic interactions with both SKI-606 and imatinib, Y253 and E255 make electrostatic interactions stabilizing the inactive conformation of Abl nucleotide binding loop, thus affecting in a more pronounced way the binding of imatinib. Similar considerations have been made for the D276 residue affecting a-Helix C inactive conformation. The extended profile of activity performed here allows us to conclude that SKI-606 is a fairly active and specific inhibitor, with a very limited number of targets outside the Abl and Src families of TK.
Abstract LBA-2 The SETBP1 gene codes for a predominantly nuclear protein with a predicted MW of 1... more Abstract LBA-2 The SETBP1 gene codes for a predominantly nuclear protein with a predicted MW of 170 kD. Germline mutations of SETBP1 were described in patients affected by the Schinzel-Giedion syndrome (SGS), a rare disease characterized by bone, muscle and cardiac abnormalities, and presenting neuroepithelial neoplasms. In an effort to investigate the molecular pathogenesis of myeloid malignancies we applied a HTS strategy, including both exome sequencing and RNA-SEQ, to atypical Chronic Myeloid Leukemia (aCML), as defined by WHO criteria, with the aim of identifying novel recurrent driver mutations. aCML shares clinical and laboratory features with CML, but it lacks the pathognomonic BCR-ABL1fusion. Since no specific recurrent genomic or karyotypic abnormalities have been identified in aCML, the molecular pathogenesis of this disease has remained elusive and the outcome dismal (median survival 37 months) with no improvement over the last 20 years. This sharply contrasts with the outcome for CML, for which the prognosis was dramatically improved by the development of imatinib as a specific inhibitor of the BCR/ABL protein. Whole-exome sequencing of 9 aCML patients revealed the presence of 62 unique mutations (range 5–14 per patient), including a recurrent alteration of SETBP1 (G870S and D868N) in three cases. Targeted resequencing performed in 70 aCMLs, 574 patients with different hematological malignancies and 344 cell lines, identified SETBP1 mutations in 17 of 70 aCML patients (24.3%; 95% CI: 16–35%), 4 of 30 (13%) MDS/MPN-u and 3 of 82 (3.6%) CMML patients. Patients with mutations had higher white blood cell counts (p=0.008) and worse prognosis (p=0.01) when tested in multivariate analysis. TF1 cells transfected with SETBP1G870S showed increased SET levels, decreased PP2A activity and increased proliferation rates. The vast majority of mutations (85%) was located between residues 858 and 871, in the SKI homologous region of SETBP1, and were identical to germline changes seen in patients with SGS. This region may be critical for ubiquitin binding and for subsequent protein degradation, since the Eukaryotic Linear Motif (ELM) identified with high probability score a putative functional site (aa. 868–873) for beta-TrCP, the substrate recognition subunit of the E3 ubiquitin ligase. This prediction was experimentally validated using biotinylated, phosphorylated peptides encompassing this region (aa 859–879): while the wild type peptide could efficiently bind beta-TrCP as predicted, a peptide presenting the G870S mutation was incapable of binding this E3 ligase subunit, indicating a possible alteration in SETBP1 protein stability caused by this mutation. In agreement with these findings, cells transfected with SETBP1G870Sshowed increased levels of SETBP1 protein when compared to cells with similar expression levels of the wild type gene. Finally, RNA-SEQ yielded gene expression profiles with overrepresentation of genes under the control of Transforming Growth Factor Beta 1 (TGFβ1) among genes differentially expressed between SETBP1-mutated and unmutated aCML patients. Mutated SETBP1 represents a novel type of oncogene which is specifically present in aCML and closely related diseases. These data allow for a better understanding of the molecular pathogenesis of this disease; they provide evidence that SETBP1 mutations might be a new biomarker for future diagnosis and classification of aCML and related diseases, and indicate a potential strategy to develop new treatment modalities for malignancies caused by mutated SETBP1. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.
Background Longitudinal single-cell sequencing experiments of patient-derived models are increasi... more Background Longitudinal single-cell sequencing experiments of patient-derived models are increasingly employed to investigate cancer evolution. In this context, robust computational methods are needed to properly exploit the mutational profiles of single cells generated via variant calling, in order to reconstruct the evolutionary history of a tumor and characterize the impact of therapeutic strategies, such as the administration of drugs. To this end, we have recently developed the LACE framework for the Longitudinal Analysis of Cancer Evolution. Results The LACE 2.0 release aimed at inferring longitudinal clonal trees enhances the original framework with new key functionalities: an improved data management for preprocessing of standard variant calling data, a reworked inference engine, and direct connection to public databases. Conclusions All of this is accessible through a new and interactive Shiny R graphical interface offering the possibility to apply filters helpful in discri...
Background Ulcerative colitis (UC) is a chronic inflammatory disorder with an unknown etiology1. ... more Background Ulcerative colitis (UC) is a chronic inflammatory disorder with an unknown etiology1. Over recent years, a growing body of evidence has been pinpointing gut virome dysbiosis as a fundamental component in its progression2, although its role during the early phases of chronic inflammation is far from being fully defined. Therefore, we sought to investigate the role of a virome-associated protein encoded by the Orthohepadnavirus genus, the Hepatitis B virus X protein (HBx)3, during UC aetiopathogenesis. Methods HBx positivity of UC patient-derived blood and gut mucosa was assessed by RT-PCR and Sanger sequencing correlated with clinical characteristics by multivariate analysis. Transcriptomics was performed on HBx-overexpressing endoscopic biopsies from healthy donors. C57BL/6 mice underwent intramucosal injections of liposome-conjugated HBx-encoding plasmids or the control. Multidimensional flow cytometry analysis was performed on colonic samples from HBx-treated and contro...
We present a large-scale analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)... more We present a large-scale analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) substitutions, considering 1,585,456 high-quality raw sequencing samples, aimed at investigating the existence and quantifying the effect of mutational processes causing mutations in SARS-CoV-2 genomes when interacting with the human host. As a result, we confirmed the presence of three well-differentiated mutational processes likely ruled by reactive oxygen species (ROS), apolipoprotein B editing complex (APOBEC), and adenosine deaminase acting on RNA (ADAR). We then evaluated the activity of these mutational processes in different continental groups, showing that some samples from Africa present a significantly higher number of substitutions, most likely due to higher APOBEC activity. We finally analyzed the activity of mutational processes across different SARS-CoV-2 variants, and we found a significantly lower number of mutations attributable to APOBEC activity in samples assigned t...
We outline the features of the R package SparseSignatures and its application to determine the si... more We outline the features of the R package SparseSignatures and its application to determine the signatures contributing to mutation profiles of tumor samples. We describe installation details and illustrate a step-by-step approach to (1) pre- pare the data for signature analysis, (2) determine the optimal parameters, and (3) employ them to determine the signatures and related exposure levels in the point mutation dataset. For complete details on the use and execution of this protocol, please refer to Lal et al. (2021).
Neoplastic cells are characterized by genetic instability, leading to the random accumulation of ... more Neoplastic cells are characterized by genetic instability, leading to the random accumulation of mutations in different tumor subpopulations, which therefore respond differently to treatments. Given the average mutational rate of tumors, the probability to acquire resistance to a single drug usually exceeds the number of cells present at diagnosis, making drug resistance a fact in most cases. Statistically, the chance of a cell being resistant to two simultaneous treatments is much lower, suggesting that drug combinations may help prevent resistance. Anaplastic large cell lymphoma (ALCL) is an aggressive type of Non-Hodgkin Lymphoma, characterized by expression of the NPM/ALK oncogenic fusion tyrosine kinase. ALK+ ALCL patients resistant or relapsed (R/R) to front-line chemotherapy have a very poor prognosis. The therapeutic activity of the ALK inhibitor crizotinib has been shown in patients with R/R ALK+ ALCL. However, 30-40% of patients develop resistance to crizotinib monotherapy...
Many large national and transnational studies have been dedicated to the analysis of SARS-CoV-2 g... more Many large national and transnational studies have been dedicated to the analysis of SARS-CoV-2 genome, most of which focused on missense and nonsense mutations. However, approximately 30% of the SARS-CoV-2 variants are synonymous, therefore changing the target codon without affecting the corresponding protein sequence.By performing a large-scale analysis of sequencing data generated from almost 400,000 SARS-CoV-2 samples, we show that silent mutations increasing the similarity of viral codons to the human ones tend to fixate in the viral genome over-time. This indicates that SARS-CoV-2 codon usage is adapting to the human host, likely improving its effectiveness in using the human aminoacyl-tRNA set through the accumulation of deceitfully neutral silent mutations.One-Sentence SummarySynonymous SARS-CoV-2 mutations related to the activity of different mutational processes may positively impact viral evolution by increasing its adaptation to human codon usage.
In the definition of fruitful strategies to contrast the worldwide diffusion of SARS-CoV-2, maxim... more In the definition of fruitful strategies to contrast the worldwide diffusion of SARS-CoV-2, maximum efforts must be devoted to the early detection of dangerous variants. An effective help to this end is granted by the analysis of deep sequencing data of viral samples, which are typically discarded after the creation of consensus sequences. Indeed, only with deep sequencing data it is possible to identify intra-host low-frequency mutations, which are a direct footprint of mutational processes that may eventually lead to the origination of functionally advantageous variants. Accordingly, a timely and statistically robust identification of such mutations might inform political decision-making with significant anticipation with respect to standard analyses based on con-sensus sequences.To support our claim, we here present the largest study to date of SARS-CoV-2 deep sequencing data, which involves 220,788 high quality samples, collected over 20 months from 137 distinct studies. Importa...
The presence of thousands of repetitive sequences makes the centromere a fragile region subject t... more The presence of thousands of repetitive sequences makes the centromere a fragile region subject to breakage. In this study we collected 31 cases of rearrangements of chromosome 18, of which 16 involved an acrocentric chromosome, during genetic screening done in three centers. We noticed a significant enrichment of reciprocal translocations between the centromere of chromosome 18 and the centromeric or pericentromeric regions of the acrocentrics. We describe five cases with translocation between chromosome 18 and an acrocentric chromosome, and one case involving the common telomere regions of chromosomes 18p and 22p. In addition, we bring evidence to support the hypothesis that chromosome 18 preferentially recombines with acrocentrics: (i) the presence on 18p11.21 of segmental duplications highly homologous to acrocentrics, that can justify a NAHR mechanism; (ii) the observation by 2D-FISH of the behavior of the centromeric regions of 18 respect to the centromeric regions of acrocent...
DOCK8 deficiency is a combined immunodeficiency due to biallelic variants in dedicator of cytokin... more DOCK8 deficiency is a combined immunodeficiency due to biallelic variants in dedicator of cytokinesis 8 (DOCK8) gene. The disease has a wide clinical spectrum encompassing recurrent infections (candidiasis, viral and bacterial infections), virally driven malignancies and immune dysregulatory features, including autoimmune (cytopenia and vasculitis) as well as allergic disorders (eczema, asthma, and food allergy). Hypomorphic function and somatic reversion of DOCK8 has been reported to result in incomplete phenotype without IgE overproduction. Here we describe a case of DOCK8 deficiency in a 8-year-old Caucasian girl. The patient’s disease was initially classified as autoimmune thrombocytopenia, which then evolved toward a combined immunodeficiency phenotype with recurrent infections, persistent EBV infection and lymphoproliferation. Two novel variants (one deletion and one premature stop codon) were characterized, resulting in markedly reduced, but not absent, DOCK8 expression. Soma...
We describe the procedures to perform the following: (1) the de novo discovery of mutational sign... more We describe the procedures to perform the following: (1) the de novo discovery of mutational signatures from raw sequencing data of viral samples and (2) the association of existing viral mutational signatures to the samples of a given dataset. The goal is to identify and characterize the nucleotide substitution patterns related to the mutational processes that underlie the origination of variants in viral genomes. The VirMutSig protocol is available at this link: https://github.com/BIMIB-DISCo/VirMutSig. For complete information on the theoretical aspects of this protocol, please refer to Graudenzi et al. (2021).
Colorectal malignancies are a leading cause of cancer death. Despite large-scale genomic efforts,... more Colorectal malignancies are a leading cause of cancer death. Despite large-scale genomic efforts, DNA mutations do not fully explain malignant evolution. Here we study the co-evolution of the genome and epigenome of colorectal tumours at single-clone resolution using spatial multi-omic profiling of individual glands. We collected 1,373 samples from 30 primary cancers and 9 concomitant adenomas and generated 1,212 chromatin accessibility profiles, 527 whole-genomes and 297 whole-transcriptomes. We found positive selection for DNA mutations in chromatin modifier genes and recurrent chromatin changes in regulatory regions of cancer drivers with otherwise no mutation. Genome-wide alterations in transcription factor binding accessibility involved CTCF, downregulation of interferon, and increased accessibility for SOX and HOX, indicating developmental genes reactivation. Epigenetic aberrations were heritable, distinguishing adenomas from cancers. Mutational signature analysis showed the e...
ObjectivesUlcerative colitis (UC) is a chronic inflammatory disorder of unknown aetiology. Gut vi... more ObjectivesUlcerative colitis (UC) is a chronic inflammatory disorder of unknown aetiology. Gut virome dysbiosis is fundamental in UC progression, although its role in the early phases of the disease is far from fully understood. Therefore, we sought to investigate the role of a virome-associated protein encoded by theOrthohepadnavirusgenus, the hepatitis B virus X protein (HBx), in UC aetiopathogenesis.DesignHBx positivity of UC patient-derived blood and gut mucosa was assessed by RT-PCR and Sanger sequencing and correlated with clinical characteristics by multivariate analysis. Transcriptomics was performed on HBx-overexpressing endoscopic biopsies from healthy donors.C57BL/6 mice underwent intramucosal injections of liposome-conjugated HBx-encoding plasmids or the control, with or without antibiotic treatment. Multidimensional flow cytometry analysis was performed on colonic samples from HBx-treated and control animals. Transepithelial electrical resistance measurement, proliferat...
Resistance to imatinib represents an important scientific and clinical issue in CML and Ph+ ALL a... more Resistance to imatinib represents an important scientific and clinical issue in CML and Ph+ ALL and is usually due to the selection of cells harbouring kinase point mutations of the BCR-ABL gene or presenting BCR-ABL gene amplification. In order to improve Bcr-Abl inhibition, several inhibitors with higher potency compared to imatinib have been identified including dasatinib and nilotinib, which show clinical activity in patients resistant to imatinib. In the present study the effects of the novel inhibitor SKI-606 on various models of resistance to imatinib were studied. SKI-606 is a selective dual Src/Abl inhibitor potently active against several CML cell lines and transfectants with IC50 values in the low nanomolar range, 2 logs lower than those obtained with imatinib. Cells expressing activated forms of c-KIT (HMC1560 and GIST882) or PDGFR (Ba/F3 Tel-PDGFRβ) were unaffected by SKI-606, in contrast to imatinib and dasatinib. SKI-606 reduced proliferation rate of cells where resistance to imatinib was caused by BCR-ABL gene amplification, such as Lama84R and of imatinib-resistant K562R and KCL22R in which no known mechanism have been identified. D276G, Y253F and E255K kinase point mutants were affected by SKI-606, resulting in inhibition of cell proliferation with IC50 values of 25, 40 and 394 nM (imatinib:1147, 1888 and 3174 nM). No activity was observed against T315I mutant. These results were confirmed in in vivo experiments, in models where resistance was not caused by mutations, as well as in cells carrying the Y253F, E255K and D276G mutations. In the first one SKI-606 was able to eradicate tumors, while significant delay in tumor growth was observed using Ba/F3 transfectants. Modelling considerations attribute the superior activity of SKI-606 to its ability to bind the intermediate (1M52) conformation of Bcr-Abl, in contrast to imatinib. Excluding the T315 residue involved in direct hydrophilic interactions with both SKI-606 and imatinib, Y253 and E255 make electrostatic interactions stabilizing the inactive conformation of Abl nucleotide binding loop, thus affecting in a more pronounced way the binding of imatinib. Similar considerations have been made for the D276 residue affecting a-Helix C inactive conformation. The extended profile of activity performed here allows us to conclude that SKI-606 is a fairly active and specific inhibitor, with a very limited number of targets outside the Abl and Src families of TK.
Abstract LBA-2 The SETBP1 gene codes for a predominantly nuclear protein with a predicted MW of 1... more Abstract LBA-2 The SETBP1 gene codes for a predominantly nuclear protein with a predicted MW of 170 kD. Germline mutations of SETBP1 were described in patients affected by the Schinzel-Giedion syndrome (SGS), a rare disease characterized by bone, muscle and cardiac abnormalities, and presenting neuroepithelial neoplasms. In an effort to investigate the molecular pathogenesis of myeloid malignancies we applied a HTS strategy, including both exome sequencing and RNA-SEQ, to atypical Chronic Myeloid Leukemia (aCML), as defined by WHO criteria, with the aim of identifying novel recurrent driver mutations. aCML shares clinical and laboratory features with CML, but it lacks the pathognomonic BCR-ABL1fusion. Since no specific recurrent genomic or karyotypic abnormalities have been identified in aCML, the molecular pathogenesis of this disease has remained elusive and the outcome dismal (median survival 37 months) with no improvement over the last 20 years. This sharply contrasts with the outcome for CML, for which the prognosis was dramatically improved by the development of imatinib as a specific inhibitor of the BCR/ABL protein. Whole-exome sequencing of 9 aCML patients revealed the presence of 62 unique mutations (range 5–14 per patient), including a recurrent alteration of SETBP1 (G870S and D868N) in three cases. Targeted resequencing performed in 70 aCMLs, 574 patients with different hematological malignancies and 344 cell lines, identified SETBP1 mutations in 17 of 70 aCML patients (24.3%; 95% CI: 16–35%), 4 of 30 (13%) MDS/MPN-u and 3 of 82 (3.6%) CMML patients. Patients with mutations had higher white blood cell counts (p=0.008) and worse prognosis (p=0.01) when tested in multivariate analysis. TF1 cells transfected with SETBP1G870S showed increased SET levels, decreased PP2A activity and increased proliferation rates. The vast majority of mutations (85%) was located between residues 858 and 871, in the SKI homologous region of SETBP1, and were identical to germline changes seen in patients with SGS. This region may be critical for ubiquitin binding and for subsequent protein degradation, since the Eukaryotic Linear Motif (ELM) identified with high probability score a putative functional site (aa. 868–873) for beta-TrCP, the substrate recognition subunit of the E3 ubiquitin ligase. This prediction was experimentally validated using biotinylated, phosphorylated peptides encompassing this region (aa 859–879): while the wild type peptide could efficiently bind beta-TrCP as predicted, a peptide presenting the G870S mutation was incapable of binding this E3 ligase subunit, indicating a possible alteration in SETBP1 protein stability caused by this mutation. In agreement with these findings, cells transfected with SETBP1G870Sshowed increased levels of SETBP1 protein when compared to cells with similar expression levels of the wild type gene. Finally, RNA-SEQ yielded gene expression profiles with overrepresentation of genes under the control of Transforming Growth Factor Beta 1 (TGFβ1) among genes differentially expressed between SETBP1-mutated and unmutated aCML patients. Mutated SETBP1 represents a novel type of oncogene which is specifically present in aCML and closely related diseases. These data allow for a better understanding of the molecular pathogenesis of this disease; they provide evidence that SETBP1 mutations might be a new biomarker for future diagnosis and classification of aCML and related diseases, and indicate a potential strategy to develop new treatment modalities for malignancies caused by mutated SETBP1. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.
Background Longitudinal single-cell sequencing experiments of patient-derived models are increasi... more Background Longitudinal single-cell sequencing experiments of patient-derived models are increasingly employed to investigate cancer evolution. In this context, robust computational methods are needed to properly exploit the mutational profiles of single cells generated via variant calling, in order to reconstruct the evolutionary history of a tumor and characterize the impact of therapeutic strategies, such as the administration of drugs. To this end, we have recently developed the LACE framework for the Longitudinal Analysis of Cancer Evolution. Results The LACE 2.0 release aimed at inferring longitudinal clonal trees enhances the original framework with new key functionalities: an improved data management for preprocessing of standard variant calling data, a reworked inference engine, and direct connection to public databases. Conclusions All of this is accessible through a new and interactive Shiny R graphical interface offering the possibility to apply filters helpful in discri...
Background Ulcerative colitis (UC) is a chronic inflammatory disorder with an unknown etiology1. ... more Background Ulcerative colitis (UC) is a chronic inflammatory disorder with an unknown etiology1. Over recent years, a growing body of evidence has been pinpointing gut virome dysbiosis as a fundamental component in its progression2, although its role during the early phases of chronic inflammation is far from being fully defined. Therefore, we sought to investigate the role of a virome-associated protein encoded by the Orthohepadnavirus genus, the Hepatitis B virus X protein (HBx)3, during UC aetiopathogenesis. Methods HBx positivity of UC patient-derived blood and gut mucosa was assessed by RT-PCR and Sanger sequencing correlated with clinical characteristics by multivariate analysis. Transcriptomics was performed on HBx-overexpressing endoscopic biopsies from healthy donors. C57BL/6 mice underwent intramucosal injections of liposome-conjugated HBx-encoding plasmids or the control. Multidimensional flow cytometry analysis was performed on colonic samples from HBx-treated and contro...
We present a large-scale analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)... more We present a large-scale analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) substitutions, considering 1,585,456 high-quality raw sequencing samples, aimed at investigating the existence and quantifying the effect of mutational processes causing mutations in SARS-CoV-2 genomes when interacting with the human host. As a result, we confirmed the presence of three well-differentiated mutational processes likely ruled by reactive oxygen species (ROS), apolipoprotein B editing complex (APOBEC), and adenosine deaminase acting on RNA (ADAR). We then evaluated the activity of these mutational processes in different continental groups, showing that some samples from Africa present a significantly higher number of substitutions, most likely due to higher APOBEC activity. We finally analyzed the activity of mutational processes across different SARS-CoV-2 variants, and we found a significantly lower number of mutations attributable to APOBEC activity in samples assigned t...
We outline the features of the R package SparseSignatures and its application to determine the si... more We outline the features of the R package SparseSignatures and its application to determine the signatures contributing to mutation profiles of tumor samples. We describe installation details and illustrate a step-by-step approach to (1) pre- pare the data for signature analysis, (2) determine the optimal parameters, and (3) employ them to determine the signatures and related exposure levels in the point mutation dataset. For complete details on the use and execution of this protocol, please refer to Lal et al. (2021).
Neoplastic cells are characterized by genetic instability, leading to the random accumulation of ... more Neoplastic cells are characterized by genetic instability, leading to the random accumulation of mutations in different tumor subpopulations, which therefore respond differently to treatments. Given the average mutational rate of tumors, the probability to acquire resistance to a single drug usually exceeds the number of cells present at diagnosis, making drug resistance a fact in most cases. Statistically, the chance of a cell being resistant to two simultaneous treatments is much lower, suggesting that drug combinations may help prevent resistance. Anaplastic large cell lymphoma (ALCL) is an aggressive type of Non-Hodgkin Lymphoma, characterized by expression of the NPM/ALK oncogenic fusion tyrosine kinase. ALK+ ALCL patients resistant or relapsed (R/R) to front-line chemotherapy have a very poor prognosis. The therapeutic activity of the ALK inhibitor crizotinib has been shown in patients with R/R ALK+ ALCL. However, 30-40% of patients develop resistance to crizotinib monotherapy...
Many large national and transnational studies have been dedicated to the analysis of SARS-CoV-2 g... more Many large national and transnational studies have been dedicated to the analysis of SARS-CoV-2 genome, most of which focused on missense and nonsense mutations. However, approximately 30% of the SARS-CoV-2 variants are synonymous, therefore changing the target codon without affecting the corresponding protein sequence.By performing a large-scale analysis of sequencing data generated from almost 400,000 SARS-CoV-2 samples, we show that silent mutations increasing the similarity of viral codons to the human ones tend to fixate in the viral genome over-time. This indicates that SARS-CoV-2 codon usage is adapting to the human host, likely improving its effectiveness in using the human aminoacyl-tRNA set through the accumulation of deceitfully neutral silent mutations.One-Sentence SummarySynonymous SARS-CoV-2 mutations related to the activity of different mutational processes may positively impact viral evolution by increasing its adaptation to human codon usage.
In the definition of fruitful strategies to contrast the worldwide diffusion of SARS-CoV-2, maxim... more In the definition of fruitful strategies to contrast the worldwide diffusion of SARS-CoV-2, maximum efforts must be devoted to the early detection of dangerous variants. An effective help to this end is granted by the analysis of deep sequencing data of viral samples, which are typically discarded after the creation of consensus sequences. Indeed, only with deep sequencing data it is possible to identify intra-host low-frequency mutations, which are a direct footprint of mutational processes that may eventually lead to the origination of functionally advantageous variants. Accordingly, a timely and statistically robust identification of such mutations might inform political decision-making with significant anticipation with respect to standard analyses based on con-sensus sequences.To support our claim, we here present the largest study to date of SARS-CoV-2 deep sequencing data, which involves 220,788 high quality samples, collected over 20 months from 137 distinct studies. Importa...
The presence of thousands of repetitive sequences makes the centromere a fragile region subject t... more The presence of thousands of repetitive sequences makes the centromere a fragile region subject to breakage. In this study we collected 31 cases of rearrangements of chromosome 18, of which 16 involved an acrocentric chromosome, during genetic screening done in three centers. We noticed a significant enrichment of reciprocal translocations between the centromere of chromosome 18 and the centromeric or pericentromeric regions of the acrocentrics. We describe five cases with translocation between chromosome 18 and an acrocentric chromosome, and one case involving the common telomere regions of chromosomes 18p and 22p. In addition, we bring evidence to support the hypothesis that chromosome 18 preferentially recombines with acrocentrics: (i) the presence on 18p11.21 of segmental duplications highly homologous to acrocentrics, that can justify a NAHR mechanism; (ii) the observation by 2D-FISH of the behavior of the centromeric regions of 18 respect to the centromeric regions of acrocent...
DOCK8 deficiency is a combined immunodeficiency due to biallelic variants in dedicator of cytokin... more DOCK8 deficiency is a combined immunodeficiency due to biallelic variants in dedicator of cytokinesis 8 (DOCK8) gene. The disease has a wide clinical spectrum encompassing recurrent infections (candidiasis, viral and bacterial infections), virally driven malignancies and immune dysregulatory features, including autoimmune (cytopenia and vasculitis) as well as allergic disorders (eczema, asthma, and food allergy). Hypomorphic function and somatic reversion of DOCK8 has been reported to result in incomplete phenotype without IgE overproduction. Here we describe a case of DOCK8 deficiency in a 8-year-old Caucasian girl. The patient’s disease was initially classified as autoimmune thrombocytopenia, which then evolved toward a combined immunodeficiency phenotype with recurrent infections, persistent EBV infection and lymphoproliferation. Two novel variants (one deletion and one premature stop codon) were characterized, resulting in markedly reduced, but not absent, DOCK8 expression. Soma...
We describe the procedures to perform the following: (1) the de novo discovery of mutational sign... more We describe the procedures to perform the following: (1) the de novo discovery of mutational signatures from raw sequencing data of viral samples and (2) the association of existing viral mutational signatures to the samples of a given dataset. The goal is to identify and characterize the nucleotide substitution patterns related to the mutational processes that underlie the origination of variants in viral genomes. The VirMutSig protocol is available at this link: https://github.com/BIMIB-DISCo/VirMutSig. For complete information on the theoretical aspects of this protocol, please refer to Graudenzi et al. (2021).
Colorectal malignancies are a leading cause of cancer death. Despite large-scale genomic efforts,... more Colorectal malignancies are a leading cause of cancer death. Despite large-scale genomic efforts, DNA mutations do not fully explain malignant evolution. Here we study the co-evolution of the genome and epigenome of colorectal tumours at single-clone resolution using spatial multi-omic profiling of individual glands. We collected 1,373 samples from 30 primary cancers and 9 concomitant adenomas and generated 1,212 chromatin accessibility profiles, 527 whole-genomes and 297 whole-transcriptomes. We found positive selection for DNA mutations in chromatin modifier genes and recurrent chromatin changes in regulatory regions of cancer drivers with otherwise no mutation. Genome-wide alterations in transcription factor binding accessibility involved CTCF, downregulation of interferon, and increased accessibility for SOX and HOX, indicating developmental genes reactivation. Epigenetic aberrations were heritable, distinguishing adenomas from cancers. Mutational signature analysis showed the e...
The complicated, evolving landscape of cancer mutations poses a formidable challenge to identify ... more The complicated, evolving landscape of cancer mutations poses a formidable challenge to identify cancer genes among the large lists of mutations typically generated in NGS experiments. The ability to prioritize these variants is therefore of paramount importance. To address this issue we developed OncoScore, a text-mining tool that ranks genes according to their association with cancer, based on available biomedical literature. Receiver operating characteristic curve and the area under the curve (AUC) metrics on manually curated datasets confirmed the excellent discriminating capability of OncoScore (OncoScore cut-off threshold = 21.09; AUC = 90.3%, 95% CI: 88.1-92.5%), indicating that OncoScore provides useful results in cases where an efficient prioritization of cancer-associated genes is needed.
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Papers by Rocco Piazza