Abstract Introduction: Anaplastic Lymphoma Kinase (ALK) tyrosine kinase inhibitors (TKIs) have dramatically improved outcomes for patients with ALK-rearranged non-small cell lung cancer (NSCLC) and other ALK-positive (ALK+) cancer types. However, the near-universal emergence of resistance underscores an urgent need for new, durable therapeutic strategies. Leveraging our prior identification of the ALK-derived peptide RPRPSQPSSL presented by HLA-B*07:02 (PMID: 37430060), we aimed to develop ALK-specific TCR-engineered T cells (ALK.TCR-T) that can selectively recognize and eliminate ALK-driven tumors. Methods: HLA-B*07:02 transgenic mice were vaccinated with the RPRPSQPSSL peptide. After two priming and two booster injections, CD137+/CD8+ T cells were sorted and subjected to single-cell sequencing for TCR discovery. The most expanded TCR clonotypes were then cloned and retrovirally transduced into human T cells to generate ALK.TCR-T cells. The anti-tumor activity of ALK.TCR-T cells was evaluated against various models of ALK+ tumors both in vitro and in vivo. Results: From twelve dominant clonotypes, six ALK-specific TCRs were identified. Two (ALK.TCR-1 and ALK.TCR-2) demonstrated >95% ALK-dextramer binding. Notably, ALK.TCR-2 bound the ALK-dextramer also in CD4+ T cells, indicating co-receptor-independent peptide-MHC recognition. Both TCRs mediated robust, antigen-specific cytotoxicity against ALK+/HLA-B*07:02+ cell lines, with no activity against mismatched targets. Engineering either EML4;ALK variant 3 or HLA-B*07:02 into non-expressing cells restored potent killing, confirming strict peptide-MHC specificity. In vivo, a single infusion of ALK.TCR-1 or ALK.TCR-2 markedly suppressed tumor growth and extended survival in a metastatic ALK+/HLA-B*07:02+ NSCLC model. Treated animals exhibited near-complete tumor clearance by day 7 post-ALK.TCR-T infusion, while control mice experienced progressive disease. Remarkably, in a metastatic ALK+/HLA-B*07:02+ neuroblastoma model, 70% (7/10) of mice achieved complete, durable responses (>100 days tumor-free) without evidence of on-target/off-tumor or off-target toxicity. Conclusion: We report the first generation of ALK-specific TCR-T cells targeting the naturally presented ALK peptide RPRPSQPSSL in the context of HLA-B*07:02. ALK.TCR-T cells display potency, selectivity, and safety across multiple ALK-driven tumor models, providing a strong rationale for advancing ALK.TCR-T cell therapy into clinical development. Citation Format: Carmen Mecca, Simone Piane, Nirmala Tilija Pun, Ana Azambuja, Luca Alessandrí, Rafael Blasco, Chi Nguyen Puc Bao, Elisa Bergaggio, Gabriele Saccu, Alessandro Gasparetto, Haley Ohlson, David A. Barbie, Ellis L. Reinherz, Marcos Simoes-Costa, Roberto Chiarle. First-in-class anaplastic lymphoma kinase (ALK)-specific TCR-T cells induce potent and selective antitumor immunity across ALK-driven human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5619.

Bioinformatics analyses depend on external software that itself relies on further programs, or dependencies, each with its own release cycle. Because tools are updated to fix bugs and add features, identical code can produce different results depending on which versions are installed, and incomplete documentation of dependencies and versions therefore undermines reproducibility. Containerization packages software with all its dependencies, but demands technical expertise and lacks a standard operating procedure. Existing workflow managers and container tools have improved reproducibility, yet each works differently and requires specialized knowledge, forcing developers to rebuild equivalent solutions from scratch. Here we present FairFlow, a framework that makes rigorous, reproducible analysis accessible without specialized technical knowledge. Developers describe a pipeline once in a declarative, INI-style specification file written in Baryon, and FairFlow automatically generates ready-to-run interfaces for R, Python, Bash, Galaxy, Nextflow and StreamFlow, each executing the analysis inside a version-locked container. The generated interfaces are self-contained: end users need only Docker and their preferred language. As a proof of concept we reanalyzed a published study in two ways: following the original protocol, which does not account for dependency installation, and with a FairFlow-based script. The first yielded only ∼21% concordance with the original findings, traced to an unspecified sequence-alignment tool version, whereas the FairFlow-based reanalysis achieved 98.8–100% concordance across operating systems and reduced setup from months of troubleshooting to a single Docker installation.

Abstract Background Achieving FAIR-compliant computational research in bioinformatics is systematically undermined by two compounding challenges that existing tools leave unresolved: long-term reproducibility and accessibility. Standard package managers re-download dependencies from live repositories at every build, making environments vulnerable to library disappearance and version drift, and pinning a package version does not pin the versions of its transitive dependencies, causing divergences between builds performed at different points in time. Compounding this, packages from repositories such as CRAN, Bioconductor, and PyPI frequently omit critical system-level dependencies from their installation metadata, leaving users to manually discover which underlying library is missing or which version is required. Beyond these technical failures, constructing a truly reproducible environment demands expertise in containerization making reproducibility in practice a privilege and not a standard. Findings We present REBEL (Reproducible Environment Builder for Explicit Library Resolution), a framework that addresses both challenges through three dependency inference heuristics: (i) Deep Inspection of source code, (ii) Fuzzy Matching against a manually curated knowledge base, and (iii) Conservative Dependency Locking. The resolved dependency stack is then archived into a self-contained local store, enabling offline and deterministic rebuilds at any future time. We compared the installation of 1,000 randomly sampled CRAN packages in isolated Docker containers versus the standard package manager and REBEL resolved 149 of 328 standard installation failures (45.4%). Moreover through its DockerBuilder component, REBEL further generates fully reproducible Docker images from a plain text requirements file, making deterministic environment construction accessible without expertise in containerization. Conclusions REBEL provides a practical foundation for FAIR-compliant, long-term reproducible bioinformatics analyses, making deterministic environment construction accessible to researchers regardless of their technical background. REBEL is freely available at https://github.com/Rebel-Project-Core

The salt production site of Piscina Torta (Rome, Italy) presents a complex chronological challenge due to its ceramic assemblage, which is almost exclusively represented by coarse ware jars with limited morphological variability. Furthermore, its occupation period coincides with the Hallstatt plateau, which hampers radiocarbon dating resolution. This study addresses these limitations by applying archaeomagnetic analyses to a kiln and ceramic fragments both from Area 1 of the site. Archaeomagnetic dating based on directions from the kiln and on archaeointensity from pottery sherds yielded two-time intervals: 930-755 BCE and 740-700 BCE. These results confirm the potential of this method as a viable dating tool, especially for problematic radiocarbon periods. At Piscina Torta, the integration of archaeomagnetic, typo- chronological, and radiocarbon data enabled the subdivision of Area 1 into three occupational phases: Phase A (725-710 BCE), Phase B (710-550 BCE), and Phase C (550-525 BCE). This study demonstrates the effectiveness of archaeomagnetism in enhancing chronological resolution in early first-millennium BCE contexts and highlights its important contribution when combined with traditional dating methods.

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Abstract Introduction: Anaplastic Lymphoma Kinase (ALK)-positive Anaplastic Large Cell Lymphoma (ALCL) is a rare subtype of T-cell lymphoma driven by nucleophosmin 1 (NPM1)-ALK fusion protein. Treatment with standard chemotherapy or ALK tyrosine kinase inhibitor crizotinib is highly effective; however, a significant portion of patients still experience relapses or refractory disease, highlighting the need for innovative therapeutic options. Our group has recently identified two ALK-specific T cell receptors targeting the human ALK peptide RPRPSQPSSL presented by HLA-B*07:02 (B7) and demonstrated specific and robust anti-tumor activity of ALK.TCR-T cells (ALK.TCR-T) in ALK+ non-small cell lung cancer [Mecca et al, Cancer res, 2024]. In this work, we aimed to address the efficacy of ALK.TCR-T in multiple models of ALK+ ALCL. Methods: Two ALK-specific TCRs were retrovirally transduced into human CD3+ T cells to generate ALK.TCR-T1 and ALK.TCR-T2. The anti-tumor activity of ALK.TCR-T was tested both in vitro and in vivo against a panel of crizotinib-sensitive and crizotinib-resistant ALK+ ALCL models. The specificity of peptide-MHC recognition was evaluated by employing ALK+/B7+, ALK+/B7-, and ALK-/B7+ cells. For in vivo studies, NSG mice were injected intravenously with ALK+ ALCL cell lines, and, after engraftment, treated with ALK.TCR-T, alone or in combination with crizotinib. Mice received 50mg/kg crizotinib by oral gavage for 10 days. Tumor growth was evaluated weekly by bioluminescence imaging. Results: In vitro killing assays demonstrated that both ALK.TCR-T selectively recognize and eliminate 80-100% of ALK+/B7+ ALCL, while no killing occurred in ALK+/B7- or ALK-/B7+ models, confirming that ALK.TCR-T specifically target the ALK peptide RPRPSQPSSL presented by HLA-B*07:02. Moreover, ALK-TCR-T were equally effective in killing crizotinib-resistant ALCL cells, independently of the mechanism driving the resistance to crizotinib. Interestingly, the combination of ALK.TCR-T and crizotinib potentiated the killing of crizotinib-sensitive ALK+ ALCL even at unfavorable E:T ratios (1:5 and 1:10).A single treatment with ALK.TCR-T significantly slowed tumor growth in an ALK+/B7+ systemic tumor model and increased the survival of mice, compared to treatment with irrelevant TCR-T cells. The combined treatment with ALK.TCR-T and crizotinib resulted in a further enhancement of tumor regression and mouse survival, with 50% (5/10) mice with no evidence of tumor 40 days after ALK.TCR-T injection. Conclusions: We demonstrated that ALK.TCR-T show specific and potent anti-tumor activity against multiple ALK+ ALCL models both in vitro and in vivo. The combination of ALK.TCR-T and crizotinib further potentiate the ability of ALK.TCR-T to control tumor growth and extend the survival of mice. These results lay the basis for developing a novel immunotherapy strategy for patients with ALK+ ALCL. Citation Format: Simone Piane, Carmen Mecca, Nirmala Tilija Pun, Ana Azambuja, Luca Alessandri, Phuc Bao Nguyen, Elisa Bergaggio, Gabriele Saccu, Alessandro Gasparetto, Haley Ohlson, Claudia Voena, Marcos Simoes-Costa, Roberto Chiarle. ALK-specific TCR-T cells showed potent and specific activity in ALK-positive anaplastic large cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5615.

This study presents new evidence for the early use of lime mortar during the Early Iron Age at the Piscina Torta site (Ostia, Italy), situated on the earliest Holocene beach ridges in the southern Tiber delta. The site, which was earlier described as a briquetage site, dates from between the late 8th and 6th century BCE and consists of a large complex of dumps, holding kilns and working floors. Lime mortar-like materials were abundantly present in these dumps and used in the construction of some kilns and working floors. To assess their nature, truly lime-based mortars or natural indurated marl, we performed petrographic and geochemical analyses on these materials, and on lime mortars experimentally produced using local marl from the Piscina Torta palaeochannel, which is the only nearby source of high-carbonate sediment in this part of the Tiber delta. The experiments showed that the marl begins to calcine effectively above 800 degrees C, developing characteristic lime mortar features between 900 degrees C and 1100 degrees C. Some archaeological materials indeed showed diagnostic microscopic features of lime mortar, including micritic binder, lime lumps, reaction rims and lack of birefringence, demonstrating that the marl had undergone the complete lime cycle (calcination, slaking and carbonation). Moreover, the analyses confirmed the origin of the archaeological mortar-like materials, being these marls. Our study provides the first evidence to date for the intentional production and use of lime mortar in central Italy, predating the widespread use of this technology in the Roman world. The results also imply that the Piscina Torta site was a multifunctional production site with a variety of kilns and activities, underscoring the technological sophistication and multipurpose nature of the site.

Functional genomics screens in human induced pluripotent stem cells (hiPSCs) remain challenging despite their transformative potential. We developed iPS2-seq: an inducible, clone-aware screening platform that enables phenotype-agnostic, single-cell resolved dissection of loss-of-function effects in hiPSC derivatives, including complex multicellular models such as organoids. iPS2-seq distinguishes true perturbation effects from genetic and epigenetic variability. It supports pooled and arrayed formats, integrates with microfluidic or split-pool single-cell RNA sequencing, and extends to multi-omic profiling of chromatin and proteins. A dedicated pipeline, catcheR, streamlines design and analysis. The platform enables stage-specific follow-up dissection of screen hits. We demonstrate this by targeting congenital heart disease-associated genes in monolayer cardiomyocytes and organoids. This reveals that epigenetic neuroectodermal priming interferes with germ layer differentiation in specific clones. Accounting for this bias, we show that SMAD2 controls cardiac progenitor specification, with knockdown redirecting cells toward fibroblast and epicardial fates. iPS2-seq unlocks rigorous functional genomics in hiPSC-based models.

Chromosomal translocations are key genomic events that frequently occur in hematopoietic cancers and solid tumors. In lymphoma and leukemia, such as diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL), most DSBs that lead to oncogenic translocation are initiated by activation-induced cytidine deaminase (AID). AID is a B-cell-specific enzyme that targets immunoglobulin (Ig) genes, considered AID on-target genes, to initiate somatic hypermutation (SHM) and class switch recombination (CSR). However, AID also exhibits off-target activity at non-immunoglobulin regions, contributing to genomic instability by promoting oncogenic chromosomal translocations and mutations that drive the development and progression of B cell lymphomas and leukemias. The mechanisms that govern AID's selective targeting of a limited subset of genomic regions remain poorly understood and represent a longstanding, fundamental question in the field. By utilizing high-throughput genome-wide translocation sequencing (HTGTS) in B cells, we previously demonstrated that PI3Kδ inhibition upregulates AID expression, thereby increasing genomic instability and partially elucidating these mechanisms. To further delineate the mechanistic cascade of AID activity as a cytidine deaminase, we now developed dU-seq to map genome-wide AID-induced mutations and applied sBLISS to detect genome-wide AID-induced DNA double-strand breaks (DSBs) in both primary B cells and CH12F3 cells. By these approaches, we successfully captured AID-mediated mutations and AID-dependent DSBs at most known translocation hotspots in the whole genome, including AID on-targets and off-targets, validating the technical robustness of our approach. Surprisingly, we identified thousands of previously unrecognized AID-mediated mutations and DSB hotspots besides those known AID target regions, revealing a much broader genomic footprint of AID activity than previously appreciated. To provide a potential clinical relevance of these discovery approaches, we tested whether emerging epigenetic therapies could impact the patterns and distribution of AID-mediated translocations in B cell lymphoma. EZH2 inhibitors, such as tazemetostat and valemetostat, are epigenetic regulators approved by the FDA for the treatment of follicular lymphoma and adult T-cell leukemia/lymphoma, respectively. We found that EZH2 inhibitors alone did not significantly alter AID expression or AID-mediated translocation frequency in CH12F3 mouse B cells or MEC-1 human B cells. However, when combined with PI3Kδ inhibition, EZH2 inhibition markedly enhanced the frequency of chromosomal translocations compared to either treatment alone in both cell models. EZH2 inhibition also further enhanced translocation formation in mouse B cells that were DNA repair deficient, such as Ligase4 knock-out cells. Mechanistically, EZH2 inhibition in B cells depletes the repressive histone modification H3K27me3 while concurrently enhancing the active histone modification H3K27ac, thereby selectively increasing transcriptional activity and facilitating chromosomal translocation formation in the presence of high AID activity or Ligase4 deficiency. Overall, to our knowledge this work represents the most comprehensive mapping of AID-induced mutational and genotoxic activity, shedding light on the whole trajectory of AID activity from the very early initiation steps of cytidine deamination to the formation of DSB intermediates up to the final outcome of chromosomal translocations. The described approach can be exploited to functionally dissect the impact of novel drugs on AID-mediated genomic instability in B cell lymphoma.

Abstract Plants have always represented a key element in landscape delineation. Indeed, plant diversity, whose distribution is influenced by geographic/climatic variability, has affected both environmental and human ecology. The present contribution represents a multi‐proxy study focused on the detection of starch, pollen and non‐pollen palynomorphs in ancient dental calculus collected from pre‐historical individuals buried at La Sassa and Pila archaeological sites (Central Italy). The collected record suggested the potential use of plant taxa by the people living in Central Italy during the Copper‐Middle Bronze Age and expanded the body of evidence reported by previous palynological and palaeoecological studies. The application of a microscopic approach provided information about domesticated crops and/or gathered wild plants and inferred considerations on ancient environments, water sources, and past health and diseases. Moreover, the research supplied data to define the natural resources (e.g., C4‐plant intake) and the social use of the space during that period. Another important aspect was the finding of plant clues referable to woody habitats, characterised by broad‐leaved deciduous taxa and generally indicative of a warm‐temperate climate and grassy vegetation. Other unusual records (e.g., diatoms, brachysclereids) participated in defining the prehistoric ecological framework. Thus, this work provides an overview on the potential of the human dental calculus analysis to delineate some features of the ancient plant ecology and biodiversity.

Abstract Introduction: Anaplastic Lymphoma Kinase (ALK) tyrosine kinase inhibitors (TKIs) have extended the survival of patients with ALK-rearranged cancers, including non-small cell lung cancer (NSCLC). Unfortunately, acquired resistance develops within 2-3 years, highlighting the urgent need for novel and effective therapeutic strategies for these patients. Here, we aimed to identify T cell receptor (TCR) clonotypes against two human ALK immunogenic peptides we previously identified by mass spectrometry in biopsies from patients with ALK-rearranged NSCLC (PMID:37430060) and to develop TCR-T cell therapies for ALK-positive NSCLCs. Methods: BALB/c mice were vaccinated with the murine ALK peptide PGPGRVAKI and transgenic mice expressing human HLA-B*07:02 were vaccinated with human ALK peptides RPRPSQPSSL and IVRCIGVSL. All mice received two priming injections (days 1 and 14) followed by two boosters, and activated CD8+ T cells were sorted and subjected to single-cell sequencing. Results: As proof of concept, we first vaccinated BALB/c mice with the ALK peptide PGPGRVAKI and identified 381 unique ALK-specific clonotypes. Among these, we cloned the most expanded 5 clonotypes and confirmed that these clonotypes were functional as evidenced by the release of significant amounts of IFN-γ and IL2, and potent in vitro killing activity following co-culture with murine ALK-positive lung cancer cells. To identify TCR clonotypes for the development of TCR-T therapy in patients with ALK-positive cancers, we next vaccinated transgenic mice expressing human HLA-B*07:02 with human ALK peptides RPRPSQPSSL and IVRCIGVSL. We again identified multiple unique ALK-specific TCR clonotypes among CD8+/CD137+ cells from these transgenic mice vaccinated with RPRPSQPSSL peptide (N=353 TCR clonotypes) and IVRCIGVSL peptide (N=742 TCR clonotypes). Single-cell RNA sequencing of the expanded clonotypes (TCR clonotype frequency ≥4) versus non-expanded clonotypes (TCR clonotype frequency <4) revealed significant upregulation of Ccl3, Ccl4, Ccl1, Ifng, Xcl1, and Il13 across mice vaccinated with RPRPSQPSSL and IVRCIGVSL (p <0.05). Gene set enrichment analysis (GSEA) confirmed significant upregulation of multiple pathways of adaptive immune response, including T cell activation, IFN-γ signaling and production, cytokine release, and T cell proliferation (adjusted p <0.05), suggesting functional activity of these TCR clonotypes against ALK. Conclusion: Here, we first demonstrated the feasibility of isolating ALK-specific TCR clonotypes by mice vaccination that exerted anti-tumor activity. Additionally, we identified ALK-specific TCR clonotypes from transgenic mice expressing human HLA-B*07:02 vaccinated with two human ALK peptides. This discovery lays the basis for the successful development of TCR-T cell therapies for ALK-positive NSCLCs, and possibly other ALK-positive cancers. Citation Format: Carmen Mecca, Ana Azambuja, Luca Alessandrí, Elisa Bergaggio, Simone Piane, Marcos Simoes-Costa, Ellis L. Reinherz, Rafael Blasco-Patiño, Roberto Chiarle. Discovery of ALK-specific TCR clonotypes for the development of TCR-T cell therapies against ALK-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 21.

Abstract Colorectal cancer (CRC) is one of the most diagnosed cancers worldwide. Though several treatment strategies are available today, around a million people die of this disease each year worldwide. Thus, there is still a pivotal need for new and more effective treatments. Genetic and non-genetic intra-tumor heterogeneity has emerged lately as an intrinsic feature of malignancies and as one of the factors responsible for therapy resistance or tolerance. In particular, the nature and role that phenotypically distinct, yet genetically indistinguishable, subpopulations have on tumor biology is far from being understood. Here we investigate phenotypic heterogeneity in CRC by identifying specific transcriptionally well defined cell subpopulations. We aim at deciphering the interactions between populations in terms of differentiation and plasticity, tumor growth and potentially exposed targetable vulnerabilities. To this end, we used patient-derived metastatic CRC (mCRC) organoids, including KRAS wild type (wt) and mutant (kras) models. We performed in total scRNAseq for 7 mCRC samples (4 wt; 3 kras) in different growth media: normal growth medium (supplemented with EGF) (n=1); medium without EGF (noEGF) (n=7); or treated with Cetuximab (CTX, a clinically approved monoclonal antibody against EGFR) (n=7). We found that the wt sample grown in EGF presents 2 distinct subpopulations: one with stemness characteristics (LGR5 high) and the other with a more differentiated phenotype (KRT20 high), in agreement with the current bibliography. Interestingly, in EGF deprived cultures new subpopulations appear. In particular, we were able to identify a subpopulation with distinct expression of WNT-related genes (APCDD1, WNT6, WIF1). This subpopulation is also present, and in cases increasing in fraction, when organoids are treated with CTX (in 3 out of 7 samples). Interestingly, a fraction of the WNT positive subpopulation seems to be able to proliferate under CTX treatment (Ki67 positive). We identified the negative regulator of the WNT pathway, APCDD1, as a potential marker to track this subpopulation. We took one wt and one kras sample as models to validate these findings by flow cytometry. Furthermore, sorting of APCDD1 positive and negative cells, and further RT-qPCR analysis, confirmed good correlation between antibody staining and gene expression. Moreover, we found that both types of cells are able to re-grow as organoids after re-culturing. This suggests that this WNT-associated subpopulation could be contributing, at least in part, to CTX tolerance or resistance. Our results show promising potential to help unravel basic features of intra-tumor heterogeneity both in basal conditions and upon treatment, and possibly to find new therapeutic targets. Citation Format: Sabrina J. Fletcher, Irene Catalano, Elena Grassi, Sofia Borgato, Barbara Peracino, Elena Piretto, Luca Alessandri, Raffaele Calogero, Luca Primo, Livio Trusolino, Andrea Bertotti, Alberto Puliafito. Unravelling intra-tumor phenotypic heterogeneity in colorectal cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6946.

The landscape characteristics and salt production methods along the coasts of peninsular Italy from the Bronze Age to the Roman period are examined, with a focus on the significance of marine salt for Italy's ancient food economy, given the limited availability of rock salt. Two main methods of marine salt production are highlighted. Briquetage involves using clay or pottery containers to evaporate seawater, which requires specific environmental conditions such as clay availability and significant human labour and technological skill. Conversely, salterns consist of creating shallow ponds or pools for seawater evaporation, demanding flat coastal areas, a stable climate, and controlled seawater flow. While this method is less labour-intensive, it requires substantial initial infrastructure.The potential for salt production in Italy's coastal palaeoenvironments using either method has been evaluated. Key factors include coastal geomorphology, climate stability, and access to raw materials. This environmental data supports an analysis of archaeological remains from salt production sites active from the Bronze Age to the Roman period. The assessment focuses on the material evidence such as clay containers from briquetage and structural remains of salterns, aiming to understand the spatial and temporal distribution of these sites.The discussion also explores the geopolitical implications of salt production, examining how the distribution and development of these sites were affected by broader geopolitical shifts in Italy, including the impact of (proto)urbanization on the expansion and adaptation of salt production practices.

In this work, we study salt-production settlement in central Italy with an exploratory application of centrality indexes, common in social network analysis: betweenness centrality, closeness centrality, and degree centrality. These methods are not new, but they have never been applied to this type of site and the results are innovative and illuminating. In fact, the closeness and degree centrality do not yield particularly interesting results. However, the betweenness centrality, which indicates the most commonly used routes in a given region, provide powerful insights. By indicating shifting most common routes through time, from the terrestrial and sea route along the coast in the Bronze and Iron Age, to the use of the Tiber River and Tiber valley as route, in the Orientalizing and Archaic Period, they allow us to advance hypotheses about the shift between two different productions. The briquetage salt production technique was used in the Bronze and Iron Age on the costal sites, which was also the most common route used in the region. While the proper marine production at the mouth of the Tiber, both on the Etruscan and Latin side, might develop during the Orientalizing and Archaic Age, together with an intensified use of the Via Salaria, running from the coast to the mountains of Latium, along the Tiber River. It would be interesting to confirm these hypotheses with further analyses and also targeted excavations.

Single-cell RNA sequencing (scRNA-seq) has emerged as a vital tool in tumour research, enabling the exploration of molecular complexities at the individual cell level. It offers new technical possibilities for advancing tumour research with the potential to yield significant breakthroughs. However, deciphering meaningful insights from scRNA-seq data poses challenges, particularly in cell annotation and tumour subpopulation identification. Efficient algorithms are therefore needed to unravel the intricate biological processes of cancer. To address these challenges, benchmarking datasets are essential to validate bioinformatics methodologies for analysing single-cell omics in oncology. Here, we present a 10XGenomics scRNA-seq experiment, providing a controlled heterogeneous environment using lung cancer cell lines characterised by the expression of seven different driver genes (EGFR, ALK, MET, ERBB2, KRAS, BRAF, ROS1), leading to partially overlapping functional pathways. Our dataset provides a comprehensive framework for the development and validation of methodologies for analysing cancer heterogeneity by means of scRNA-seq.

Abstract Introduction: Approximately 50% of human cancers have somatic integrations of long interspersed element-1 (LINE-1; also known as L1). L1 is a retrotransposon that copies itself through RNA and integrates into new genomic loci. Although L1 sequences comprise approximately 17% of the human genome, only 100-150 L1 loci have retained the ability to retrotranspose. These full-length, retrotransposition-competent L1s are typically repressed in human somatic cells, but there is evidence of their significant reactivation in human cancers. Therefore, L1 overexpression is a hallmark of many human cancers, particularly in highly malignant tumor types such as breast, ovarian, pancreatic, esophageal, lung, head-and-neck, colon, prostate, and liver cancers. Results: In many cancers, chromosomal translocations are fundamental pathogenetic events initiated by the generation of DNA double-strand breaks (DSBs). Although LINE-1 (L1) retrotransposition events have been implicated in chromosomal rearrangements associated with most human cancers, the frequency and mechanisms by which L1 is involved in these rearrangements, including chromosomal translocations, remain poorly characterized. By combining high-throughput genome-wide translocation sequencing (HTGTS) and two sequencing techniques to detect L1 insertion sites (PolyA-seq and CELTICS-seq), we demonstrated that the presence of an active human L1 retrotransposition significantly increases the frequency of chromosomal translocations. By identifying L1-dependent translocation hotspots in HEK293T cells we highlight the requirement for the L1 reverse transcriptase (RT) activity and the crucial role of pre-existing DNA DSBs in promoting L1-mediated chromosomal translocations. L1-dependent translocation hotspots showed a marked preference for transcription end sites (TES) of active genes, early replication timing regions, and accessible chromatin regions. Therefore, while cancer genome analysis provides a static view of chromosomal rearrangements based on canonical L1 integrations, our functional studies reveal an unanticipated predominance of chromosomal translocations facilitated by non-canonical L1 retrotransposition via RNA-templated repair of existing DSBs, largely expanding our knowledge on the mechanism by which L1 retrotransposition induces genomic instability in human cancers. Citation Format: Jianli Tao, Luca Alessandri, Carlos Mendez-Dorantes, Kathleen H. Burns, Qi Wang, Roberto Chiarle. LINE-1 retrotransposition promotes chromosomal translocations through RNA-templated DNA repair in human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 404.