Innovative paraffin embedding to unlock archival blocks for highly demanding genomic analyses

Innovative paraffin embedding to unlock archival blocks for highly demanding genomic analyses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Innovative paraffin embedding to unlock archival blocks for highly demanding genomic analyses Enrico Berrino, Enrico Berrino, Sara Bellomo, Anita Chesta, Raffella Giorgio, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8787286/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract DNA analysis from formalin-fixed paraffin-embedded (FFPE) tissues is frequently compromised by fragmentation and fixation-induced artifacts, limiting advanced genomic applications. While fixation chemistry has been extensively studied, the contribution of paraffin embedding to DNA damage remains poorly defined. Here, starting from a direct experimental observation of embedding-associated DNA degradation, we identify paraffin embedding as an unrecognized source of oxidative DNA damage that can be mitigated by supplementing paraffin with lipophilic antioxidants. We analysed 190 samples spanning pre-clinical and clinical settings, including 126 murine specimens and 64 human tumors processed using four fixation and embedding workflows. Using more than 180 sequencing assays — including targeted sequencing, whole-exome sequencing, shallow whole-genome sequencing, and long-read Oxford Nanopore whole-genome sequencing — we show that antioxidant-supplemented paraffin consistently preserves DNA integrity, increasing DNA integrity number (DIN) values and enriching for DNA fragments exceeding 10 kb in length. In human tumors, antioxidant-supplemented paraffin enabled robust performance across all tested genomic applications, reducing fixation-related artifacts and improving compatibility with both short- and long-read sequencing. Collectively, these findings identify paraffin embedding as a key pre-analytical determinant of molecular preservation and provide a simple, scalable strategy for enabling genomic analysis from archival pathology specimens. Health sciences/Molecular medicine Health sciences/Oncology/Cancer/Tumour biomarkers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 5 Introduction Paraffin embedding of fixed tissues, a time-honoured technique underlying histological preparation, is practiced globally, producing millions of tissue blocks for diagnostic purposes and potential future reuse. Originally devised by Wilhelm His Sr. in 1868 1 , this procedure enables micron-thin sectioning and remains in widespread use today. While the sequence and practice of the process have remained largely unchanged over time, the scope of its application and the performance required the final product have evolved substantially. Originally developed to preserve morphology, paraffin embedding is now expected to support histochemical, immunohistochemical and molecular analyses, providing diagnostic, prognostic and therapeutic information central for personalized medicine and reflecting the increasing centrality of molecular profiling in modern precision oncology 2 . Although nucleic acids derived from fresh-frozen tissues are theoretically optimal for genomic analyses, multiple practical considerations necessitate the use of formalin-fixed paraffin-embedded (FFPE) tissue blocks in both research and clinical settings 3 , 4 . A major advantage of FFPE specimens is their universal availability, as they are routinely collected using standardized protocols and stored in dedicated archives for prolonged periods, often spanning decades. Moreover, the preservation of tissue architecture allows precise morphological and immunohistochemical assessment, allowing accurate selection of tumor regions prior to nucleic acid extraction. Importantly, FFPE tissue blocks frequently represent the only available clinical material for addressing diagnostic, prognostic and therapy-related questions. As a consequence, considerable effort has been devoted to improving molecular preservation in FFPE tissues and developing sequencing workflows specifically adapted to degraded or chemically modified nucleic acids, including next-generation sequencing (NGS)–based approaches. Nevertheless, the suboptimal quality of nucleic acids extracted from FFPE specimens remains a major limitation, often compromising both feasibility and reliability of downstream genomic analyses 5 . Multiple pre-analytical steps contribute to the generation of FFPE tissue blocks, each with the potential to affect molecular preservation 6 , 7 . Among these, fixation in neutral buffered formalin (NBF) is widely recognized as the most detrimental, inducing extensive DNA fragmentation and chemically induced base modifications 8 . Enhanced DNA preservation can be achieved using acid-depleted aldehyde fixatives, including acid-depleted formalin and acid-free glyoxal (GAF) 9 . However, even these alternatives do not preserve DNA integrity at the level required by highly demanding applications such as whole-genome sequencing (WGS), which rely on long, intact DNA molecules. Paraffin embedding, by contrast, is generally regarded as biologically inert, as reflected by the etymology of the term “paraffin” (“parum affinis”, meaning “with little affinity”). Here, we report that a systematic evaluation of FFPE processing steps revealed an unexpected contribution of paraffin embedding to nucleic acid degradation. We hypothesized that this effect may be driven by oxidative stress generated within heated paraffin and that it could be mitigated by antioxidant supplementation. To test this hypothesis, we sYstematically analyzed murine and human cells and tissues fixed in either NBF or GAF, embedded in standard or antioxidant-supplemented paraffin, and assessed DNA integrity and preservation. Results Antioxidant addition during tissue processing preserved DNA integrity in preclinical models An initial experimental observation revealed a phenomenon that guided the direction of this study (Fig. 1 A). Cancer cell lines (A549 and MCF7), fixed in NBF or GAF, without undergoing the subsequent processing steps required for paraffin embedding, displayed markedly higher DIN values compared to those embedded in tissue blocks. This first indication motivated the conceptual framework shown in Fig. 1 , prompting us to consider the molecular processes potentially responsible for DIN deterioration. Because oxidative reactions represent a major source of DNA damage, we tested whether limiting oxidation during processing would preserve DNA integrity (Fig. 1 B). To define suitable antioxidant additives, we screened a panel of liposoluble compounds for in paraffin through literature evaluation (Supplementary Table 1). Irganox 1010 and Irganox 1076 emerged as the most compatible and consistently improved nucleic acid integrity and were thus selected for downstream analyses. Therefore, four murine tissues (two kidneys and two spleens) underwent either the standard (Std) workflow or supplemented with Irganox antioxidants (AO) at each step, fixed with both NBF and GAF. DNA integrity was consistently improved in AO-treated samples (median DIN = 6.3 vs. 4.1 under standard processing, p = 0.001, Fig. 1 C), irrespective of the used fixative. We then revisited our initial model and hypothesized that the strongest oxidative pressure may occur during paraffin embedding, driven by elevated paraffin temperature, prolonged tissue exposure to atmospheric O₂, and paraffin autoxidation over time. To test this, we compared four conditions: NBF embedding in standard paraffin (F), NBF embedding in AO-supplemented paraffin (FAO), GAF embedding in standard paraffin (G), and GAF embedding in AO-supplemented paraffin (GAO). Across 14 murine tissues processed in parallel (Supplementary Table 2), DNA integrity showed a clear increasing trend (F median = 4.1, FAO median = 4.5, G median = 4.9, GAO median = 6.4), with GAF combined with AO yielding the highest DIN values across the entire cohort. Building on the superior performance of GAF-based workflows, we assessed the AO performance across an expanded set of 27 murine tissues for G and GAO protocols. The results (Supplementary Fig. 1 and Supplementary Table 3) corroborated this effect, with GAO-fixed samples yielding markedly higher DNA integrity (median DIN = 6.3) than tissues processed with the standard G protocol (median DIN = 4.3, p = 0.001). Antioxidant addition during tissue processing preserved DNA integrity in human samples The same four fixation–embedding workflows were next applied in parallel to 16 human cancers (Supplementary Table 4). Quality control metrics on the extracted DNA revealed no differences in extraction yield when stratifying samples by fixative (GAF vs NBF), processing type (Std vs AO), or complete workflow (F, FAO, G, GAO) (Supplementary Table 5). In contrast, DNA integrity was consistently higher in AO-processed than in Std-processed samples (Supplementary Table 6), and in GAF-fixed than NBF-fixed samples (Supplementary Fig. 7). Overall, human tissues mirrored the murine results: DIN values increased progressively from F-processed samples (most fragmented), through FAO and standard GAF samples (showing comparable integrity) (Fig. 2 A). These differences became even more pronounced when considering that DIN is derived from TapeStation electropherograms, which follow a logarithmic scale. Pseudotraces generated from the median electropherogram signal of all F-processed and GAO-processed samples further underscored this effect: F-fixed tissues displayed a median fragment size of 658 bp, whereas GAO-fixed tissues reached 12247 bp (Fig. 2 B). Notably, GAO samples showed that more than 70% of DNA fragments exceeded 10 kb, a proportion significantly higher than that observed with any other fixation workflow (Fig. 2 C). Most FFPE DNA extraction protocols include a high-temperature incubation (90°C) to reverse formalin crosslinks, a step unnecessary for GAF-fixed tissues where such adducts do not form. To determine whether this heating stage affects DNA integrity, we re-extracted all samples from each fixation workflow. Under the 90°C condition, all workflows yielded similarly low DIN values (F = 3.4, FAO = 3.35, G = 4.3, GAO = 3.6, Supplementary Fig. 2). We then computed the DIN variation between the first and second extraction (ΔDIN): GAO samples exhibited the greatest drop (ΔDIN median = 3), revealing a marked susceptibility of high-integrity DNA to the 90°C incubation (Fig. 2 D). Short reads, Comprehensive Genome Profiling (CGP) on human samples We next applied the clinical-grade TSOComp sequencing assay to assess the impact of the alternative fixation and embedding workflows on pre-analytical and analytical performance. No sequencing failures were observed (64/64 successful sequencing, Supplementary Table 8). Sequencing metrics—including total read counts and mean depth of coverage—were comparable across all four protocols (Supplementary Tables 5, 6 and 7). Notably, GAO-fixed tissues generated libraries with substantially longer insert sizes than any other workflow (Fig. 3 A; Supplementary Table 5). This increase in fragment length was accompanied by improved coverage uniformity, with GAO samples showing the highest and most consistent proportion of target regions exceeding 250× depth (Fig. 3 B). In line with pre-analytical findings, GAF and FAO specimens displayed moderate improvements over formalin/standard paraffin processing, which remained the least favorable condition. No additional sequencing parameters differed significantly among workflows. We then assessed variant-calling accuracy by cross-comparing sequencing outputs across fixation protocols. Four CGP-derived metrics were examined: tumor mutation burden (TMB), microsatellite instability (MSI) status, Jaccard Index (JI), and COSMIC Single Base Substitution (SBS) signatures. Detailed descriptions of these analytical parameters are provided in the Materials and Methods. TMB and MSI status were strongly correlated in our cohort (r Pearson = 0.86, p < 0.001, Supplementary Fig. 3), with three tumors displaying an MSI-high/TMB-high phenotype. Stratification by fixation protocol revealed no significant differences in either TMB or MSI status (Supplementary Table 5). Using the Jaccard Index, we quantified the degree of variant overlap across fixation protocols. As expected, patient-specific genotypes were the dominant driver of clustering (Supplementary Fig. 4). Nevertheless, fixation-based grouping revealed that only F-fixed tumors showed weak correlation in variant calling, whereas the remaining workflows were strongly concordant with one another (Fig. 3 C). To further investigate these differences, we inferred COSMIC SBS signatures using somatic variants grouped by fixation protocol. The resulting heatmap (Fig. 3 D) showed a prominent contribution of the MMR-deficiency–associated signature SBS16, consistent with the three MSI-high lesions. In contrast, SBS1 — associated with FFPE-related artifacts — was approximately two-fold higher in F-fixed samples compared with GAO-processed tissues, reflecting the increased C > T transitions characteristic of formalin-induced DNA damage; FAO and standard GAF workflows showed intermediate levels. Signature reconstructions for each group are reported in the Supplementary Fig. 5. Short reads, Whole Exome Sequencing (WES) and shallow Whole Genome Sequencing (sWGS) on human samples We next evaluated the performance of AO-based protocols in more demanding short-read sequencing applications. To this end, we applied Whole-Exome Sequencing (WES) to the two fixation workflows representing the extremes of DNA integrity (F and GAO), achieving a median depth of 100×. In parallel, we implemented shallow Whole-Genome Sequencing (sWGS) by exploiting a rapid and cost-effective strategy to assess copy-number profiles across the entire cohort. WES performance showed a striking contrast between fixation/embedding workflows. The QC p-value heatmap (Fig. 4 A, Supplementary Table 9, Supplementary Fig. 6) revealed a robust segregation of samples, with differences emerging across nearly all functional QC categories. Only total throughput metrics remained comparable while every other parameter displayed a sharp polarization between F and GAO workflows. GAO-fixed/embedded tissues consistently outperformed all other conditions, exhibiting broader coverage across depth thresholds (1×–100×), superior on-target capture, and markedly improved coverage uniformity. Conversely, F-fixed tumors showed the hallmarks of degraded template quality—including elevated off-target rates, larger fractions of uncovered regions, and reduced uniformity—underscoring the detrimental impact of formalin-derived DNA damage on exome performance. We next applied COSMIC single base substitution (SBS) signature analysis to evaluate the impact of fixation/processing protocols on variant calling. After identifying the most likely somatic variants through population- and genome-based filtering (see Materials and Methods), mutational signatures were inferred for each sample and subsequently aggregated across the F and GAO cohorts. The resulting profiles (Fig. 4 B) showed contributions from SBS1 and SBS5, together with the MMR deficiency–associated signatures SBS6 and SBS15, consistent with the MSI status of a subset of CRCs. Pairwise comparisons revealed that the formalin-associated SBS1 signature was significantly reduced in GAO-fixed/processed tumors compared with F-fixed lesions (12% vs 20%; p = 0.019). Notably, analysis of the merged cohorts identified SBS1, SBS5 and SBS6 as the dominant contributors, with SBS1 accounting for 29% of substitutions in F-fixed samples versus 16% in GAO-fixed/processed tissues, further supporting a substantial reduction of fixation/processing-induced mutational artifacts under GAO conditions (Fig. 4 C). Shallow WGS quality metrics revealed clear differences across the four fixation/processing protocol workflows (Supplementary Table 10). GAO-fixed/processed samples achieved higher sequencing depth, and a greater number of aligned bases compared with all other conditions, whereas F-fixed lesions displayed a significantly elevated soft-clipping rate, indicative of reduced read quality. We next inferred genome-wide copy-number (CN) profiles using fixed window sizes to assess potential fixation-related effects on CN estimation (Supplementary Table 11). For comparative analysis, log 2 ratio profiles were aggregated by fixation/processing protocol and correlation analyses were performed using window sizes of 500 kb, 250 kb, 100 kb and 50 kb. As shown in Supplementary Fig. 7, aside from the expected and gradual reduction in correlation when moving from larger to smaller bins, no protocol-specific differences were observed in CN profiles, indicating that CN inference remained robust across tested protocols. Pre-analytical long read sequencing, with the ONT Whole Genome Sequencing (ONT-WGS) on human samples. We next provisionally evaluated long-read whole-genome sequencing using Oxford Nanopore Technologies (ONT) on 10 paired tissues fixed and processed with either F or GAO protocols, all meeting the DNA input requirements for ONT barcoding (1 µg). The resulting 20 libraries were distributed across four flow cells. From aligned reads, we assessed three core ONT-WGS performance metrics: total reads per sample, total bases, and read length N50, which provides a base-weighted estimate of effective read length (Supplementary Table 12). No significant differences were observed in the number of total reads assigned per sample between fixation/processing protocols, indicating comparable sequencing throughput. In contrast, both total bases and read length (N50) significantly differed, reflecting a pronounced shift in read length distribution. As shown in Fig. 5 , GAO-processed samples were enriched for substantially longer reads compared with F-fixed tissues (N50 comparison: p = 0.0007, Fig. 5 A), and a higher number of total bases evaluated (Total Bases comparison, p = 0.007, Fig. 5 B). Discussion Multiple pre-analytical steps contribute to the generation of formalin-fixed paraffin-embedded (FFPE) tissue blocks, each with the potential to affect molecular preservation 10 , 11 . Among these, formalin fixation is widely recognized as the most detrimental, inducing extensive DNA fragmentation and chemically induced base modifications 12 , 13 . We and others have shown that enhanced DNA preservation can be achieved using acid-depleted aldehyde fixatives, such as acid-depleted formalin and acid-free glyoxal (GAF) 9 . However, even these alternatives do not preserve DNA integrity to the extent required for highly demanding applications such as whole-genome or whole-exome sequencing, which rely on long, intact DNA molecules. Consistent with this limitation, DNA fragmentation varies widely across FFPE tissue blocks 6 , 14 and reduced fragment length—reflected by low DNA Integrity Number (DIN) values—substantially compromises the accuracy of genetic testing and the performance of next-generation sequencing and amplicon-based assays 15 – 17 . Although recent analytical advances partially improve data recovery from FFPE material 18 , 19 , DNA fragmentation remains an intrinsic constraint of FFPE processing. In contrast to formaldehyde, glyoxal reacts differently with nucleic acids despite its similar chemical structure. Glyoxal does not form stable protein–DNA cross-links and preferentially reacts with guanine, generating unstable adducts 20 . We previously introduced acid-free glyoxal (GAF) as a non-toxic histological fixative 21 , and an international validation study confirmed that GAF preserves tissue morphology and antigenicity comparably to formalin 22 . Importantly, GAF fixation results in significantly improved DNA preservation compared with NBF 9 . Unexpectedly, comparative analyses of murine tissues and cultured cells before and after paraffin embedding revealed a marked increase in DNA fragmentation following embedding, irrespective of fixation time. This finding challenges the long-standing assumption that paraffin — chemically inert — is biologically inactive, revealing “a paraffin embedding paradox” ( PE paradox ), whereby nucleic acid degradation occurs during the embedding process itself. We reasoned that this effect is not caused by paraffin per se , but by oxygen dissolved within molten wax, which promotes oxidative DNA damage under the elevated temperatures required for embedding 23 . To counteract this oxidative stress, we developed an antioxidant-supplemented paraffin containing a defined mixture of Irganox-type antioxidants. Initial experiments in murine tissues demonstrated a significant improvement in DNA integrity relative to standard paraffin. These findings were subsequently extended to human tumors processed using four fixation and embedding workflows (F, FAO, G, GAO), revealing that the combined use of GAF fixation and antioxidant-supplemented paraffin consistently yielded the highest DNA integrity, reduced fragmentation, and fewer fixation-related mutational artifacts. Across sequencing platforms, DNA fragment length—rather than bulk yield—emerged as the primary determinant of analytical performance. This is consistent with reports showing that FFPE-induced fragmentation directly affects library complexity and coverage uniformity 24 – 26 . In both targeted sequencing and shallow WGS (CUTseq) assays, antioxidant-based workflows produced longer insert sizes and more homogeneous coverage, potentially improving sensitivity and reproducibility 25 , 27 , 28 . Variant concordance analysis using the Jaccard Index confirmed patient-specific genotype as the dominant clustering factor but revealed reduced concordance exclusively in standard formalin/standard paraffin samples, consistent with FFPE-induced stochastic artifacts 13 , 25 , 29 . Mutational signature analysis further distinguished biological signal from technical noise: MSI-associated signatures were preserved across workflows, whereas the FFPE-associated SBS1 signature was significantly enriched in formalin/standard paraffin samples 30 , 31 and markedly reduced by antioxidant-supplemented embedding. Whole-exome sequencing showed a strong polarization of QC metrics, with GAO samples displaying superior on-target capture and coverage uniformity despite comparable throughput and other parameters critical for variant sensitivity in FFPE-based WES 26 , 28 . In contrast, shallow whole-genome sequencing for copy-number inference remained robust across workflows, consistent with the relative tolerance of CN analysis to moderate fragmentation 32 , 33 . Finally, long-read Oxford Nanopore sequencing provided a direct functional readout of high-molecular-weight DNA preservation. While total read counts were similar, GAO samples yielded significantly higher total bases and N50 values. This is particularly relevant because ONT performance depends primarily on read length rather than read number 34 – 36 . Recent Nanopore studies on FFPE tumors report median N50 values of ~ 500–600 bp 37 , underscoring how antioxidant-supplemented paraffin markedly exceeds conventional FFPE performance and expands the applicability of archival pathology material to long-read genomics. Taken together, the results of the study show that the combination of GAF fixation with AO paraffin (GAO workflow) yielded the highest level of DNA preservation observed to date in paraffin-embedded tissues. Of interest, antioxidant supplementation also partially mitigated DNA damage in formalin-fixed tissues, indicating that formaldehyde-induced lesions arise through a multistep damage process that continues during tissue processing and can be amplified by oxidative conditions 13 , 30 . In conclusion, this study identifies paraffin embedding as a previously underappreciated contributor to DNA degradation in histological specimens and shows that this damage can be mitigated by supplementing paraffin with lipophilic antioxidants. This simple modification preserves long DNA fragments and improves the performance of multiple sequencing applications in both formalin- and glyoxal-fixed tissues. Although validation in independent laboratories and larger cohorts will be required, particularly to assess low-frequency variant detection beyond depth-related limitations, antioxidant-supplemented paraffin represents a scalable strategy to potentially enable broad genomic utility of archival pathology specimens. Declarations Participant consent Written informed consent was obtained from all participants prior to inclusion in the study, in accordance with the Declaration of Helsinki and as approved by the Institutional Ethics Review Board of the Città della Salute e della Scienza Hospital of Turin (Protocol No. 0028088; Document File No. 28/2024). Acknowledgements CM discloses support for the research of this work from: FPRC 5 per mille MUR 2021 [CHI-RO], FONDAZIONE AIRC under IG 2025 [ID. 32346 project], FINPIEMONTE [P.R.F.E.S.R. 2021/27]. BB and GB disclose support from FINPIEMONTE [Piedmont Regional Program F.E.S.R. 2021/27] and MUR, Eurostars Project 2024, SCRATCH (Eurostars 4508). Competing interest: PD and CMu are employees of ADDAX Biosciences S.r.l.. BB is co-founder, and GB serves as CEO of ADDAX Biosciences S.r.l.. ADDAX Biosciences S.r.l. developed and patented GAF and AO Paraffin (“genoWax”) and provided them for the study. CM reports personal fees from an advisory board role for Roche, Illumina, AstraZeneca, Daiichi Sankyo outside the scope of the present work. Author Contributions: GB conceived the study and highlighting the PE Paradox and planning the use of antioxidants in preventing DNA fragmentation. EB designed the experiments, performed most of the experimental work, coordinated the study and wrote the manuscript. SEB performed the bioinformatic and computational analyses. AC and RG carried out wet-lab experiments. M.C. provided murine tissues. PD and CMu were responsible for the management and processing of human and murine samples. IC, FDG and AB performed sample collection. BB critically revised the manuscript. CM provided the financial support needed for the study, critically supported experimental planning and data analysis, and reviewed the first draft of the manuscript. All authors approved the final version of the manuscript. Data availability : all data are provided as supplementary material. Code availability: all codes are previously developed and cited in the text. Material and Methods Pre-clinical models Cultured human cell lines (A549, RRID:CVCL_0023, from the American Type Culture Collection and MCF-7, RRID:CVCL_0031, from NCI-60) and tissues from Balb/c mice were used for the experimental setting of the procedure. Studies on animal tissues were conducted in accordance with the national guidelines and regulations and were approved by the Italian Health Ministry (Authorization N. CC652.N.YOS). Briefly, cells and mouse tissues (spleen, kidney, liver and HER2-overexpressing mammary tumors from BALB-neuT female mice) were fixed with either Neutral Buffered Formalin (NBF, Diapath, Martinengo (BG), Italy) or Glyoxal Acid Free (GAF) (a 2% solution of Glyoxal deprived of acids in 0.1 M phosphate buffer, pH 7.4) (ADDAX Biosciences, Turin, Italy). Following fixation for 24 h at room temperature, tissues were processed for histological analysis by dehydration, clearing and paraffin embedding using two automated tissue processors. Both systems employed standard reagents for dehydration and clearing. In one processor (Leica ASP 300, Leica Microsystems, Wetzlar, Germany), tissues were embedded using standard paraffin wax (BIO-Optica, 20134 Milan, Italy) with a melting point of 56–58°C. In the second processor (Histo PRO 300, Histoline, 20090 Pantigliate, Italy), the same paraffin wax was used but supplemented with antioxidants (AO paraffin; see below). Paraffin blocks in cassettes were then prepared using alternatively standard paraffin or AO-Paraffin. Anatomical sites of origin were reported in Supplementary Table 2. Human samples Sampling was performed from 16 cases of human tumors large enough to allow multiple sampling (8 colorectal cancers, 3 renal cancers, 2 liver tumors, 1 thymoma, 1 gastric cancer and 1 ovarian cancer, Supplementary Table 4). The study and the related project were approved by the Board responsible for “Biobanking and use of human tissues for experimental studies” of the Department of Medical Sciences, University of Turin and by the Institutional Ethics Review Board of the Città della Salute e della Scienza Hospital of Turin (Protocol No. 0028088; Document File No. 28/2024). Parallel slices were fixed, two in NBF and two in GAF fixative. Following fixation at room temperature for 24 hours, NBF and the GAF-fixed tissue blocks were processed for paraffin embedding, either in the standard paraffin wax or in the AO Paraffin (see above). Overall, for each case four fixation/embedding protocols were tested: one tissue block (F) was fixed in NBF and embedded in standard paraffin; one (FAO) was fixed in NBF and embedded in paraffin supplemented with Antioxidants; one (G) was fixed in GAF and embedded in standard paraffin; one (GAO) was fixed in GAF and embedded in paraffin supplemented with Antioxidants. Analyses were therefore conducted in parallel on 64 tissue blocks. DNA Extraction DNA from fixed-only cells was extracted using the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany). For fixed and embedded tissues, 3-μm-thick sections were cut from paraffin blocks and stained for Hematoxylin-Eosin and immunohistochemistry (IHC), additional 5-μm-thick sections were used for nucleic acid (NA) extraction.DNA extraction was performed following histological assessment of cellular content on hematoxylin and eosin (H&E)–stained sections using the QIAamp DNA FFPE Tissue Kit (QIAGEN, Hilden, Germany). Unless otherwise specified, samples were processed without the 90°C incubation step typically used for formalin crosslink reversal. This high-temperature incubation was included only in dedicated experiments designed to assess the impact of heat-induced decrosslinking on DNA fragmentation. Nucleic acids were quantified using the Qubit fluorometer (Qubit, ThermoFisher Scientific, Waltham, MA, USA) and assessed for integrity with the Agilent 4150 TapeStation System (Agilent Technologies, Santa Clara, CA, US), returning the DNA Integrity Number (DIN). Targeted Next Generation Sequencing Genomic DNA purified from the 64 fixed and embedded human cancer tissues was subjected to targeted deep sequencing using the TruSight Oncology 500 panel (TSO500; Illumina, San Diego, CA, USA), which covers 523 cancer-related genes, spanning approximately 1.2 Mb of coding regions (1.94 Mb total genomic coverage). Library preparation was performed according to the manufacturer’s instructions and sequencing was carried out on an Illumina NextSeq 550 platform. Raw sequencing data were processed using the DRAGEN on-site analysis pipeline (v4.0; Illumina) in tumor-only mode, aligned to the human reference genome GRCh37 (hg19). Variants with a variant allele frequency (VAF) >5% were retained and annotated using InterVar, as previously described. Putative germline variants were bioinformatically flagged by querying population databases including gnomAD Exomes, gnomAD Genomes, and the 1000 Genomes Project, applying a minimum alternative allele count of 50 and a population allele frequency threshold of ≥0.01. Remaining variants were annotated as putative somatic. TMB and MSI were calculated as previously reported 38 . Single base substitution (SBS) mutational signatures were inferred from targeted sequencing data generated with the TruSight Oncology 500 panel for each single fixation type. Only putative somatic variants with a variant allele frequency (VAF) >5% were considered for downstream analyses in the .vcf file. Somatic variants were used as input for mutational signature assignment using SigProfilerAssignment, applying reference signature sets from COSMIC Mutational Signatures v3.5 (Human Cancer) and COSMIC Experimental Signatures v1.0. Signature attribution was performed using default parameters 39 , and the relative contribution of each SBS signature was estimated for individual samples as well as for aggregated cohorts stratified by fixation and embedding workflow. Jaccard Index was calculated as previously reported 9 . Shallow whole-genome sequencing Shallow whole-genome sequencing (sWGS) was performed on all the 64 human cancer samples using the CUTseq protocol, as previously described and validated 40-42 . Libraries were prepared following the published procedure and sequenced on an Illumina NovaSeq 6000 system using a high-output 75 bp single-end configuration. Sequencing data were processed using a custom bioinformatic pipeline. Read counts were computed in fixed genomic bins of 50, 100, 250 and 500 kb, and genome-wide copy-number profiles were generated by applying circular binary segmentation to log 2 -transformed read counts using the DNAcopy R package (v1.74.1). Pearson correlation values were calculated for each sample within the four different fixation protocols. Whole Exome Sequencing WES libraries were prepared using the Agilent SureSelect Clinical Research Exome V4 (CRE V4) (Agilent, Santa Clara, CA, USA) and sequenced on an Illumina NovaSeq platform (Illumina, San Diego, CA, USA) producing 2×150bp paired-end reads. Raw sequencing data were then aligned to human reference genome hg19 using the Burrows-Wheeler Aligner (v.0.7.17), followed by BAM files sorting and indexing via SAMtools. We used Picard tools and Genome Analysis Toolkit (GATK v4.5) for duplicate marking, base quality score recalibration and coverage metrics statistics. Somatic short variant calling was performed with Mutect2 in tumor-only mode. Variants were annotated with ANNOVAR/InterVar against the hg19 reference genome. Germline-like variants were excluded based on population allele frequencies (gnomAD, 1000 Genomes, ESP6500 ≥0.001), ClinVar benign annotations, or variant allele fraction (VAF) >0.9. Somatic-like variants were defined as rare or absent from population databases (<1×10⁻⁴), supported by high sequencing depth (DP ≥40, AD_ALT ≥5), and restricted to single-nucleotide variants. AF thresholds were set to 0.15–0.80. Variants not meeting these criteria were classified as germline proxy. For mutational signature analysis, only high-confidence somatic SNVs located in coding or splicing regions were retained. Minimal VCF files containing CHROM, POS, REF, and ALT were generated and analyzed using MutationalPatterns to derive 96-channel trinucleotide profiles 39 . Oxford Nanopore Technology (ONT) sequencing We applied ONT, long read sequencing to 10 F- and 10 GAO-fixed tumors. 500 ng from each sample were used as input DNA to prepare libraries by means of the Native Barcoding Kit 24 V14 (Oxford Nanopore Technologies plc, UK) and then sequenced with the WGS Ligation Sequencing Kit DNA V14 (Oxford Nanopore Technologies plc, UK) following the manufacturer's instructions. We sequenced the five libraries/flow cell on the PromethION P24 instrument. Demultiplexed FASTQ files were aligned to the human reference genome hg19 and the resulting .bam files analysed for the sequencing metrics using the NanoPlot tool ( github.com/wdecoster/NanoPlot ). Statistical Analysis Statistics were performed with R software v4.03. Differences in distributions were analysed with a paired t-test, and contingency was assessed using by Fisher exact test or chi-square test. Antioxidants for AO protocols A mixture of different liposoluble Anti-Oxidants of the Irganox type (Irganox 1010 (Pentaerythritol tetrakis(3,5-di- tert -butyl-4-hydroxyhydrocinnamate)), Irganox 1076 (Octadecyl 3-(3,5-di- tert -butyl-4-hydroxyphenyl)propionate), all from Sigma-Aldrich (Milan, Italy); and Irganox 1520 (2-Methyl-4,6-bis(octylsulfanylmethyl)phenol) from BASF Italia, (Cesano Maderno, MB, Italy) was dissolved in standard paraffin wax for histology. The deleterious effect of minor impurities present in standard paraffin on the performance of WGS sequencing (see below) could be prevented by the adoption of a highly purified paraffin wax. The resulting reagent, genoWax®, was prepared and supplied by Addax Biosciences srl, (Turin, Italy). Histological analysis Prior to the study, we examined the effect of adding a small amount of liposoluble antioxidants (below 0.5% w/v) to standard paraffin. Tests conducted on the technical suitability of AO paraffin (processing, sectioning), and focused on the structural features of embedded tissues (in H&E-stained sections) showed no appreciable differences, assuring that the antioxidant-supplemented paraffin could be safely adopted as an histological embedding medium. References van der Lem T, de Bakker M, Keuck G, Richardson MK (2021) Wilhelm His Sr. and the development of paraffin embedding. Pathologe 42:55–61. 10.1007/s00292-021-00947-4 Mosele MF et al (2024) Recommendations for the use of next-generation sequencing (NGS) for patients with advanced cancer in 2024: a report from the ESMO Precision Medicine Working Group. Ann Oncol 35:588–606. 10.1016/j.annonc.2024.04.005 Mathieson W, Thomas GA (2020) Why Formalin-fixed, Paraffin-embedded Biospecimens Must Be Used in Genomic Medicine: An Evidence-based Review and Conclusion. J Histochem Cytochem 68:543–552. 10.1369/0022155420945050 Blow N (2007) Tissue preparation: Tissue issues. 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Pathologica 117:423–429. 10.32074/1591-951X-N1157 Zhang N et al (2024) High clonal diversity and spatial genetic admixture in early prostate cancer and surrounding normal tissue. Nat Commun 15:3475. 10.1038/s41467-024-47664-z Simonetti M et al (2021) COVseq is a cost-effective workflow for mass-scale SARS-CoV-2 genomic surveillance. Nat Commun 12:3903. 10.1038/s41467-021-24078-9 Additional Declarations Yes there is potential Competing Interest. PD and CMu are employees of ADDAX Biosciences S.r.l.. BB is co-founder, and GB serves as CEO of ADDAX Biosciences S.r.l.. ADDAX Biosciences S.r.l. developed and patented GAF and AO Paraffin (“genoWax”) and provided them for the study. CM reports personal fees from an advisory board role for Roche, Illumina, AstraZeneca, Daiichi Sankyo outside the scope of the present work. Supplementary Files SupplementaryFigure1.pdf Supplementary Figure 1 SupplementaryFigure6.pdf Supplementary Figure 6 SupplementaryTable1.xlsx Supplementary Table 1 SupplementaryTable2.xlsx Supplementary Table 2 SupplementaryTable6.xlsx Supplementary Table 6 SupplementaryFigure7.pdf Supplementary Figure 7 SupplementaryTable10.xlsx Supplementary Table 10 SupplementaryTable3.xlsx Supplementary Table 3 SupplementaryTable7.xlsx Supplementary Table 7 SupplementaryFigure2.pdf Supplementary Figure 2 SupplementaryTable5.xlsx Supplementary Table 5 SupplementaryFigure3.pdf Supplementary Figure 3 SupplementaryTable9.xlsx Supplementary Table 9 SupplementaryTable4.xlsx Supplementary Table 4 SupplementaryTable11.xlsx Supplementary Table 11 SupplementaryTable8.xlsx Supplementary Table 8 SupplementaryFigure4.pdf Supplementary Figure 4 SupplementaryFigure5.pdf Supplementary Figure 5 SupplementaryData.docx Supplementary Data Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8787286","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":586523074,"identity":"e7b48b00-346b-4f4c-9c56-3011a0c85ac3","order_by":0,"name":"Enrico 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Italy","correspondingAuthor":false,"prefix":"","firstName":"Gianni","middleName":"","lastName":"Bussolati","suffix":""}],"badges":[],"createdAt":"2026-02-04 13:50:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8787286/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8787286/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102599929,"identity":"ec739953-23a2-4f77-93ed-10f89939ee99","added_by":"auto","created_at":"2026-02-13 12:49:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6045301,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eParaffin embedding induces oxidative DNA fragmentation. A\u003c/strong\u003e. TapeStation electropherograms and DNA Integrity Number (DIN) values from A549 and MCF7 cells fixed in neutral buffered formalin (F) or glyoxal acid-free (GAF, G), analyzed before (FF) or after paraffin embedding (FFPE). \u003cstrong\u003eB\u003c/strong\u003e. Schematic of the working hypothesis linking tissue processing to oxidative DNA damage. \u003cstrong\u003eC\u003c/strong\u003e. DIN values from murine tissues processed with standard (Std) or antioxidant-supplemented (AO) workflows. \u003cstrong\u003eD\u003c/strong\u003e. Refined model identifying paraffin embedding as a major source of oxidative DNA damage. \u003cstrong\u003eE\u003c/strong\u003e. DIN values across fixation–embedding workflows (F, FAO, G, GAO). Boxplots show median and interquartile range. ns, not significant; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/26dded479288b733a36fdb83.png"},{"id":102599917,"identity":"d2e48529-808f-4b91-aaaa-130d598917ba","added_by":"auto","created_at":"2026-02-13 12:49:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":971542,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntioxidant-supplemented paraffin (AO) preserves long DNA fragments in human tumors. A\u003c/strong\u003e. DNA Integrity Number (DIN) values from 64 human tumors processed with F, FAO, G and GAO workflows. \u003cstrong\u003eB\u003c/strong\u003e. Median TapeStation pseudoelectropherograms comparing F and GAO samples. \u003cstrong\u003eC\u003c/strong\u003e. Fraction of DNA fragments \u0026gt;10 kb across workflows. \u003cstrong\u003eD\u003c/strong\u003e. Change in DIN (ΔDIN) following 90 °C re-extraction. Boxplots indicate median and interquartile range; *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/90ed20461729262f04f74ee8.png"},{"id":102747438,"identity":"992c4c12-5a10-41d8-9699-02325472e053","added_by":"auto","created_at":"2026-02-16 09:04:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":771148,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImproved targeted sequencing performance and reduced artifacts with GAO processing. A\u003c/strong\u003e. Library insert size distributions from TruSight Oncology 500 sequencing. \u003cstrong\u003eB\u003c/strong\u003e. Percentage of target regions covered at ≥250× depth. \u003cstrong\u003eC\u003c/strong\u003e. Jaccard Index matrix showing variant concordance across workflows. \u003cstrong\u003eD\u003c/strong\u003e. Relative contribution of COSMIC single base substitution (SBS) signatures aggregated by fixation–embedding protocol.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/f0b69a797bdcb8015a95df1b.png"},{"id":102747233,"identity":"41bd4ae8-0dfa-4d03-a242-6028483cb21b","added_by":"auto","created_at":"2026-02-16 09:04:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5378774,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntioxidant-based workflows improve whole-exome sequencing quality. A.\u003c/strong\u003eHeatmap of whole-exome sequencing quality metrics comparing F and GAO workflows. \u003cstrong\u003eB\u003c/strong\u003e. COSMIC SBS signature contributions inferred from WES data. \u003cstrong\u003eC\u003c/strong\u003e. Reconstructed mutational signatures for F- and GAO-processed samples and the relative contributions across workflows.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/197895b56244aa4bb1f5b2f3.png"},{"id":103503915,"identity":"694c5b82-aae8-4fb7-a68e-cf0546941dd7","added_by":"auto","created_at":"2026-02-26 13:04:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":252640,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLong-read sequencing is enabled by antioxidant-supplemented paraffin. A\u003c/strong\u003e. Read length N50 from Oxford Nanopore whole-genome sequencing of F- and GAO-processed tumors. \u003cstrong\u003eB\u003c/strong\u003e. Total bases sequenced per sample. Boxplots show median and interquartile range; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/d1b1e0673dfea4adbabe8c14.png"},{"id":102865107,"identity":"300fcbf0-bfeb-4d0b-8825-67e3e6f14f08","added_by":"auto","created_at":"2026-02-17 16:42:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":252640,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLong-read sequencing is enabled by antioxidant-supplemented paraffin. A\u003c/strong\u003e. Read length N50 from Oxford Nanopore whole-genome sequencing of F- and GAO-processed tumors. \u003cstrong\u003eB\u003c/strong\u003e. Total bases sequenced per sample. Boxplots show median and interquartile range; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/9ababfc8a034327a7ebf9407.png"},{"id":105903828,"identity":"50de7759-49fb-4f07-a224-b13f57100878","added_by":"auto","created_at":"2026-04-01 09:54:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18317069,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/b55844c6-6583-479a-b379-56ce370e8b5b.pdf"},{"id":102747143,"identity":"2f3ba21a-2040-4746-a233-1ffa02549b44","added_by":"auto","created_at":"2026-02-16 09:04:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8177,"visible":true,"origin":"","legend":"Supplementary Figure 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1","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/754f5250df74a530707a4e0b.xlsx"},{"id":102747338,"identity":"608a9dd6-17f6-473c-adf7-9b3ef665544a","added_by":"auto","created_at":"2026-02-16 09:04:33","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":11836,"visible":true,"origin":"","legend":"Supplementary Table 2","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/fd084626f58915d5e8ea0f08.xlsx"},{"id":102746872,"identity":"d5326fbb-b3b0-4281-8884-59fe8c7d5ab8","added_by":"auto","created_at":"2026-02-16 09:02:20","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":9393,"visible":true,"origin":"","legend":"Supplementary Table 6","description":"","filename":"SupplementaryTable6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/5854d5ccc2f5d50a5cf7e6c0.xlsx"},{"id":102747431,"identity":"56ddea6c-366e-40a0-a1ad-24e2a6dbe1f0","added_by":"auto","created_at":"2026-02-16 09:04:46","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":2631690,"visible":true,"origin":"","legend":"Supplementary Figure 7","description":"","filename":"SupplementaryFigure7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/2b9dcaa6453377a529093c6b.pdf"},{"id":102599924,"identity":"26bc93f0-4104-4091-b055-4a8152902ada","added_by":"auto","created_at":"2026-02-13 12:49:34","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":28329,"visible":true,"origin":"","legend":"Supplementary Table 10","description":"","filename":"SupplementaryTable10.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/749ef70b5082a7c81ea02699.xlsx"},{"id":102747583,"identity":"4b4cafc0-57c2-45a4-8915-1979a3cafdf8","added_by":"auto","created_at":"2026-02-16 09:04:59","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":10367,"visible":true,"origin":"","legend":"Supplementary Table 3","description":"","filename":"SupplementaryTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/7e2e69e7512fef6e66cb14c5.xlsx"},{"id":102962541,"identity":"b9552b9c-0c27-4d1c-a5f0-fa11543f1299","added_by":"auto","created_at":"2026-02-19 04:09:44","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":9528,"visible":true,"origin":"","legend":"Supplementary Table 7","description":"","filename":"SupplementaryTable7.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/458ad24770ad6613b733357a.xlsx"},{"id":102599928,"identity":"8d5cf94d-27dc-46d0-b183-68462908ea24","added_by":"auto","created_at":"2026-02-13 12:49:34","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":14527,"visible":true,"origin":"","legend":"Supplementary Figure 2","description":"","filename":"SupplementaryFigure2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/e4e261f32bfa774625ad5fa2.pdf"},{"id":102747137,"identity":"7d7db64c-fbbf-4973-92c6-1915123e52d7","added_by":"auto","created_at":"2026-02-16 09:03:55","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":12063,"visible":true,"origin":"","legend":"Supplementary Table 5","description":"","filename":"SupplementaryTable5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/a26ec0a4bdabb12e9ce5d87d.xlsx"},{"id":102599925,"identity":"460408fe-e63a-4587-bf4f-3d58e5762ae0","added_by":"auto","created_at":"2026-02-13 12:49:34","extension":"pdf","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":8154,"visible":true,"origin":"","legend":"Supplementary Figure 3","description":"","filename":"SupplementaryFigure3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/bf71a830ae6596d936c663a8.pdf"},{"id":102747442,"identity":"7627b2fc-61c2-4c9c-81c6-c7116ac65687","added_by":"auto","created_at":"2026-02-16 09:04:46","extension":"xlsx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":28701,"visible":true,"origin":"","legend":"Supplementary Table 9","description":"","filename":"SupplementaryTable9.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/6f624dc73e2e1ef8d0415624.xlsx"},{"id":102748157,"identity":"6a9cf04e-33ff-4631-b349-c988fd47611f","added_by":"auto","created_at":"2026-02-16 09:06:08","extension":"xlsx","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":29584,"visible":true,"origin":"","legend":"Supplementary Table 4","description":"","filename":"SupplementaryTable4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/e1962ee27c2890e9073d5bf3.xlsx"},{"id":102599923,"identity":"e5312647-b8a6-4e8f-a664-2ffd04565cb9","added_by":"auto","created_at":"2026-02-13 12:49:33","extension":"xlsx","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":88297608,"visible":true,"origin":"","legend":"Supplementary Table 11","description":"","filename":"SupplementaryTable11.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/e90654f70e24bc34d24dd201.xlsx"},{"id":102599931,"identity":"58c34d5d-d9b7-4122-9c11-62da4b27e9c4","added_by":"auto","created_at":"2026-02-13 12:49:34","extension":"xlsx","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":22738,"visible":true,"origin":"","legend":"Supplementary Table 8","description":"","filename":"SupplementaryTable8.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/4c9536687d71ffd1a574131b.xlsx"},{"id":102599926,"identity":"7d7cabff-0278-4cb8-a8ed-cd2aa5ed7e31","added_by":"auto","created_at":"2026-02-13 12:49:34","extension":"pdf","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":19158,"visible":true,"origin":"","legend":"Supplementary Figure 4","description":"","filename":"SupplementaryFigure4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/4b7466d156faf2ecf0d58631.pdf"},{"id":102748353,"identity":"47ed4c2b-eafd-4061-8f0f-764f920ab9b5","added_by":"auto","created_at":"2026-02-16 09:10:42","extension":"pdf","order_by":18,"title":"","display":"","copyAsset":false,"role":"supplement","size":8715342,"visible":true,"origin":"","legend":"Supplementary Figure 5","description":"","filename":"SupplementaryFigure5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/af7ea528a41139f2fd665064.pdf"},{"id":102747210,"identity":"381e4651-972c-49cd-a80e-51058c511c69","added_by":"auto","created_at":"2026-02-16 09:04:10","extension":"docx","order_by":19,"title":"","display":"","copyAsset":false,"role":"supplement","size":21282,"visible":true,"origin":"","legend":"Supplementary Data","description":"","filename":"SupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-8787286/v1/a7b21faa1161dac305ca9e06.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nPD and CMu are employees of ADDAX Biosciences S.r.l.. BB is co-founder, and GB serves as CEO of ADDAX Biosciences S.r.l.. ADDAX Biosciences S.r.l. developed and patented GAF and AO Paraffin (“genoWax”) and provided them for the study. CM reports personal fees from an advisory board role for Roche, Illumina, AstraZeneca, Daiichi Sankyo outside the scope of the present work.","formattedTitle":"Innovative paraffin embedding to unlock archival blocks for highly demanding genomic analyses","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParaffin embedding of fixed tissues, a time-honoured technique underlying histological preparation, is practiced globally, producing millions of tissue blocks for diagnostic purposes and potential future reuse. Originally devised by Wilhelm His Sr. in 1868\u003csup\u003e1\u003c/sup\u003e, this procedure enables micron-thin sectioning and remains in widespread use today. While the sequence and practice of the process have remained largely unchanged over time, the scope of its application and the performance required the final product have evolved substantially. Originally developed to preserve morphology, paraffin embedding is now expected to support histochemical, immunohistochemical and molecular analyses, providing diagnostic, prognostic and therapeutic information central for personalized medicine and reflecting the increasing centrality of molecular profiling in modern precision oncology\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Although nucleic acids derived from fresh-frozen tissues are theoretically optimal for genomic analyses, multiple practical considerations necessitate the use of formalin-fixed paraffin-embedded (FFPE) tissue blocks in both research and clinical settings\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. A major advantage of FFPE specimens is their universal availability, as they are routinely collected using standardized protocols and stored in dedicated archives for prolonged periods, often spanning decades. Moreover, the preservation of tissue architecture allows precise morphological and immunohistochemical assessment, allowing accurate selection of tumor regions prior to nucleic acid extraction. Importantly, FFPE tissue blocks frequently represent the only available clinical material for addressing diagnostic, prognostic and therapy-related questions. As a consequence, considerable effort has been devoted to improving molecular preservation in FFPE tissues and developing sequencing workflows specifically adapted to degraded or chemically modified nucleic acids, including next-generation sequencing (NGS)\u0026ndash;based approaches. Nevertheless, the suboptimal quality of nucleic acids extracted from FFPE specimens remains a major limitation, often compromising both feasibility and reliability of downstream genomic analyses\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Multiple pre-analytical steps contribute to the generation of FFPE tissue blocks, each with the potential to affect molecular preservation\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Among these, fixation in neutral buffered formalin (NBF) is widely recognized as the most detrimental, inducing extensive DNA fragmentation and chemically induced base modifications\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Enhanced DNA preservation can be achieved using acid-depleted aldehyde fixatives, including acid-depleted formalin and acid-free glyoxal (GAF)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, even these alternatives do not preserve DNA integrity at the level required by highly demanding applications such as whole-genome sequencing (WGS), which rely on long, intact DNA molecules. Paraffin embedding, by contrast, is generally regarded as biologically inert, as reflected by the etymology of the term \u0026ldquo;paraffin\u0026rdquo; (\u0026ldquo;parum affinis\u0026rdquo;, meaning \u0026ldquo;with little affinity\u0026rdquo;). Here, we report that a systematic evaluation of FFPE processing steps revealed an unexpected contribution of paraffin embedding to nucleic acid degradation. We hypothesized that this effect may be driven by oxidative stress generated within heated paraffin and that it could be mitigated by antioxidant supplementation. To test this hypothesis, we sYstematically analyzed murine and human cells and tissues fixed in either NBF or GAF, embedded in standard or antioxidant-supplemented paraffin, and assessed DNA integrity and preservation.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant addition during tissue processing preserved DNA integrity in preclinical models\u003c/h2\u003e \u003cp\u003eAn initial experimental observation revealed a phenomenon that guided the direction of this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Cancer cell lines (A549 and MCF7), fixed in NBF or GAF, without undergoing the subsequent processing steps required for paraffin embedding, displayed markedly higher DIN values compared to those embedded in tissue blocks. This first indication motivated the conceptual framework shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, prompting us to consider the molecular processes potentially responsible for DIN deterioration. Because oxidative reactions represent a major source of DNA damage, we tested whether limiting oxidation during processing would preserve DNA integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). To define suitable antioxidant additives, we screened a panel of liposoluble compounds for in paraffin through literature evaluation (Supplementary Table\u0026nbsp;1). Irganox 1010 and Irganox 1076 emerged as the most compatible and consistently improved nucleic acid integrity and were thus selected for downstream analyses. Therefore, four murine tissues (two kidneys and two spleens) underwent either the standard (Std) workflow or supplemented with Irganox antioxidants (AO) at each step, fixed with both NBF and GAF. DNA integrity was consistently improved in AO-treated samples (median DIN\u0026thinsp;=\u0026thinsp;6.3 vs. 4.1 under standard processing, p\u0026thinsp;=\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), irrespective of the used fixative.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then revisited our initial model and hypothesized that the strongest oxidative pressure may occur during paraffin embedding, driven by elevated paraffin temperature, prolonged tissue exposure to atmospheric O₂, and paraffin autoxidation over time. To test this, we compared four conditions: NBF embedding in standard paraffin (F), NBF embedding in AO-supplemented paraffin (FAO), GAF embedding in standard paraffin (G), and GAF embedding in AO-supplemented paraffin (GAO). Across 14 murine tissues processed in parallel (Supplementary Table\u0026nbsp;2), DNA integrity showed a clear increasing trend (F median\u0026thinsp;=\u0026thinsp;4.1, FAO median\u0026thinsp;=\u0026thinsp;4.5, G median\u0026thinsp;=\u0026thinsp;4.9, GAO median\u0026thinsp;=\u0026thinsp;6.4), with GAF combined with AO yielding the highest DIN values across the entire cohort.\u003c/p\u003e \u003cp\u003eBuilding on the superior performance of GAF-based workflows, we assessed the AO performance across an expanded set of 27 murine tissues for G and GAO protocols. The results (Supplementary Fig.\u0026nbsp;1 and Supplementary Table\u0026nbsp;3) corroborated this effect, with GAO-fixed samples yielding markedly higher DNA integrity (median DIN\u0026thinsp;=\u0026thinsp;6.3) than tissues processed with the standard G protocol (median DIN\u0026thinsp;=\u0026thinsp;4.3, p\u0026thinsp;=\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAntioxidant addition during tissue processing preserved DNA integrity in human samples\u003c/h3\u003e\n\u003cp\u003eThe same four fixation\u0026ndash;embedding workflows were next applied in parallel to 16 human cancers (Supplementary Table\u0026nbsp;4). Quality control metrics on the extracted DNA revealed no differences in extraction yield when stratifying samples by fixative (GAF vs NBF), processing type (Std vs AO), or complete workflow (F, FAO, G, GAO) (Supplementary Table\u0026nbsp;5). In contrast, DNA integrity was consistently higher in AO-processed than in Std-processed samples (Supplementary Table\u0026nbsp;6), and in GAF-fixed than NBF-fixed samples (Supplementary Fig.\u0026nbsp;7). Overall, human tissues mirrored the murine results: DIN values increased progressively from F-processed samples (most fragmented), through FAO and standard GAF samples (showing comparable integrity) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These differences became even more pronounced when considering that DIN is derived from TapeStation electropherograms, which follow a logarithmic scale. Pseudotraces generated from the median electropherogram signal of all F-processed and GAO-processed samples further underscored this effect: F-fixed tissues displayed a median fragment size of 658 bp, whereas GAO-fixed tissues reached 12247 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Notably, GAO samples showed that more than 70% of DNA fragments exceeded 10 kb, a proportion significantly higher than that observed with any other fixation workflow (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMost FFPE DNA extraction protocols include a high-temperature incubation (90\u0026deg;C) to reverse formalin crosslinks, a step unnecessary for GAF-fixed tissues where such adducts do not form. To determine whether this heating stage affects DNA integrity, we re-extracted all samples from each fixation workflow. Under the 90\u0026deg;C condition, all workflows yielded similarly low DIN values (F\u0026thinsp;=\u0026thinsp;3.4, FAO\u0026thinsp;=\u0026thinsp;3.35, G\u0026thinsp;=\u0026thinsp;4.3, GAO\u0026thinsp;=\u0026thinsp;3.6, Supplementary Fig.\u0026nbsp;2). We then computed the DIN variation between the first and second extraction (ΔDIN): GAO samples exhibited the greatest drop (ΔDIN median\u0026thinsp;=\u0026thinsp;3), revealing a marked susceptibility of high-integrity DNA to the 90\u0026deg;C incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\n\u003ch3\u003eShort reads, Comprehensive Genome Profiling (CGP) on human samples\u003c/h3\u003e\n\u003cp\u003eWe next applied the clinical-grade TSOComp sequencing assay to assess the impact of the alternative fixation and embedding workflows on pre-analytical and analytical performance. No sequencing failures were observed (64/64 successful sequencing, Supplementary Table\u0026nbsp;8). Sequencing metrics\u0026mdash;including total read counts and mean depth of coverage\u0026mdash;were comparable across all four protocols (Supplementary Tables\u0026nbsp;5, 6 and 7). Notably, GAO-fixed tissues generated libraries with substantially longer insert sizes than any other workflow (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA; Supplementary Table\u0026nbsp;5). This increase in fragment length was accompanied by improved coverage uniformity, with GAO samples showing the highest and most consistent proportion of target regions exceeding 250\u0026times; depth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In line with pre-analytical findings, GAF and FAO specimens displayed moderate improvements over formalin/standard paraffin processing, which remained the least favorable condition. No additional sequencing parameters differed significantly among workflows.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then assessed variant-calling accuracy by cross-comparing sequencing outputs across fixation protocols. Four CGP-derived metrics were examined: tumor mutation burden (TMB), microsatellite instability (MSI) status, Jaccard Index (JI), and COSMIC Single Base Substitution (SBS) signatures. Detailed descriptions of these analytical parameters are provided in the Materials and Methods. TMB and MSI status were strongly correlated in our cohort (r Pearson\u0026thinsp;=\u0026thinsp;0.86, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Supplementary Fig.\u0026nbsp;3), with three tumors displaying an MSI-high/TMB-high phenotype. Stratification by fixation protocol revealed no significant differences in either TMB or MSI status (Supplementary Table\u0026nbsp;5). Using the Jaccard Index, we quantified the degree of variant overlap across fixation protocols. As expected, patient-specific genotypes were the dominant driver of clustering (Supplementary Fig.\u0026nbsp;4). Nevertheless, fixation-based grouping revealed that only F-fixed tumors showed weak correlation in variant calling, whereas the remaining workflows were strongly concordant with one another (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). To further investigate these differences, we inferred COSMIC SBS signatures using somatic variants grouped by fixation protocol. The resulting heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) showed a prominent contribution of the MMR-deficiency\u0026ndash;associated signature SBS16, consistent with the three MSI-high lesions. In contrast, SBS1 \u0026mdash; associated with FFPE-related artifacts \u0026mdash; was approximately two-fold higher in F-fixed samples compared with GAO-processed tissues, reflecting the increased C\u0026thinsp;\u0026gt;\u0026thinsp;T transitions characteristic of formalin-induced DNA damage; FAO and standard GAF workflows showed intermediate levels. Signature reconstructions for each group are reported in the Supplementary Fig.\u0026nbsp;5.\u003c/p\u003e\n\u003ch3\u003eShort reads, Whole Exome Sequencing (WES) and shallow Whole Genome Sequencing (sWGS) on human samples\u003c/h3\u003e\n\u003cp\u003eWe next evaluated the performance of AO-based protocols in more demanding short-read sequencing applications. To this end, we applied Whole-Exome Sequencing (WES) to the two fixation workflows representing the extremes of DNA integrity (F and GAO), achieving a median depth of 100\u0026times;. In parallel, we implemented shallow Whole-Genome Sequencing (sWGS) by exploiting a rapid and cost-effective strategy to assess copy-number profiles across the entire cohort.\u003c/p\u003e \u003cp\u003eWES performance showed a striking contrast between fixation/embedding workflows. The QC p-value heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Supplementary Table\u0026nbsp;9, Supplementary Fig.\u0026nbsp;6) revealed a robust segregation of samples, with differences emerging across nearly all functional QC categories. Only total throughput metrics remained comparable while every other parameter displayed a sharp polarization between F and GAO workflows. GAO-fixed/embedded tissues consistently outperformed all other conditions, exhibiting broader coverage across depth thresholds (1\u0026times;\u0026ndash;100\u0026times;), superior on-target capture, and markedly improved coverage uniformity. Conversely, F-fixed tumors showed the hallmarks of degraded template quality\u0026mdash;including elevated off-target rates, larger fractions of uncovered regions, and reduced uniformity\u0026mdash;underscoring the detrimental impact of formalin-derived DNA damage on exome performance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next applied COSMIC single base substitution (SBS) signature analysis to evaluate the impact of fixation/processing protocols on variant calling. After identifying the most likely somatic variants through population- and genome-based filtering (see Materials and Methods), mutational signatures were inferred for each sample and subsequently aggregated across the F and GAO cohorts. The resulting profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) showed contributions from SBS1 and SBS5, together with the MMR deficiency\u0026ndash;associated signatures SBS6 and SBS15, consistent with the MSI status of a subset of CRCs. Pairwise comparisons revealed that the formalin-associated SBS1 signature was significantly reduced in GAO-fixed/processed tumors compared with F-fixed lesions (12% vs 20%; p\u0026thinsp;=\u0026thinsp;0.019). Notably, analysis of the merged cohorts identified SBS1, SBS5 and SBS6 as the dominant contributors, with SBS1 accounting for 29% of substitutions in F-fixed samples versus 16% in GAO-fixed/processed tissues, further supporting a substantial reduction of fixation/processing-induced mutational artifacts under GAO conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eShallow WGS quality metrics revealed clear differences across the four fixation/processing protocol workflows (Supplementary Table\u0026nbsp;10). GAO-fixed/processed samples achieved higher sequencing depth, and a greater number of aligned bases compared with all other conditions, whereas F-fixed lesions displayed a significantly elevated soft-clipping rate, indicative of reduced read quality. We next inferred genome-wide copy-number (CN) profiles using fixed window sizes to assess potential fixation-related effects on CN estimation (Supplementary Table\u0026nbsp;11). For comparative analysis, log\u003csub\u003e2\u003c/sub\u003e ratio profiles were aggregated by fixation/processing protocol and correlation analyses were performed using window sizes of 500 kb, 250 kb, 100 kb and 50 kb. As shown in Supplementary Fig.\u0026nbsp;7, aside from the expected and gradual reduction in correlation when moving from larger to smaller bins, no protocol-specific differences were observed in CN profiles, indicating that CN inference remained robust across tested protocols.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePre-analytical long read sequencing, with the ONT Whole Genome Sequencing (ONT-WGS) on human samples.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe next provisionally evaluated long-read whole-genome sequencing using Oxford Nanopore Technologies (ONT) on 10 paired tissues fixed and processed with either F or GAO protocols, all meeting the DNA input requirements for ONT barcoding (1 \u0026micro;g). The resulting 20 libraries were distributed across four flow cells. From aligned reads, we assessed three core ONT-WGS performance metrics: total reads per sample, total bases, and read length N50, which provides a base-weighted estimate of effective read length (Supplementary Table\u0026nbsp;12). No significant differences were observed in the number of total reads assigned \u003cem\u003eper\u003c/em\u003e sample between fixation/processing protocols, indicating comparable sequencing throughput. In contrast, both total bases and read length (N50) significantly differed, reflecting a pronounced shift in read length distribution. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, GAO-processed samples were enriched for substantially longer reads compared with F-fixed tissues (N50 comparison: p\u0026thinsp;=\u0026thinsp;0.0007, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), and a higher number of total bases evaluated (Total Bases comparison, p\u0026thinsp;=\u0026thinsp;0.007, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMultiple pre-analytical steps contribute to the generation of formalin-fixed paraffin-embedded (FFPE) tissue blocks, each with the potential to affect molecular preservation\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Among these, formalin fixation is widely recognized as the most detrimental, inducing extensive DNA fragmentation and chemically induced base modifications\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. We and others have shown that enhanced DNA preservation can be achieved using acid-depleted aldehyde fixatives, such as acid-depleted formalin and acid-free glyoxal (GAF)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, even these alternatives do not preserve DNA integrity to the extent required for highly demanding applications such as whole-genome or whole-exome sequencing, which rely on long, intact DNA molecules.\u003c/p\u003e \u003cp\u003eConsistent with this limitation, DNA fragmentation varies widely across FFPE tissue blocks\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e and reduced fragment length\u0026mdash;reflected by low DNA Integrity Number (DIN) values\u0026mdash;substantially compromises the accuracy of genetic testing and the performance of next-generation sequencing and amplicon-based assays\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Although recent analytical advances partially improve data recovery from FFPE material\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, DNA fragmentation remains an intrinsic constraint of FFPE processing.\u003c/p\u003e \u003cp\u003eIn contrast to formaldehyde, glyoxal reacts differently with nucleic acids despite its similar chemical structure. Glyoxal does not form stable protein\u0026ndash;DNA cross-links and preferentially reacts with guanine, generating unstable adducts\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. We previously introduced acid-free glyoxal (GAF) as a non-toxic histological fixative\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, and an international validation study confirmed that GAF preserves tissue morphology and antigenicity comparably to formalin\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Importantly, GAF fixation results in significantly improved DNA preservation compared with NBF\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUnexpectedly, comparative analyses of murine tissues and cultured cells before and after paraffin embedding revealed a marked increase in DNA fragmentation following embedding, irrespective of fixation time. This finding challenges the long-standing assumption that paraffin \u0026mdash; chemically inert \u0026mdash; is biologically inactive, revealing \u0026ldquo;a \u003cem\u003eparaffin embedding paradox\u0026rdquo;\u003c/em\u003e (\u003cem\u003ePE paradox\u003c/em\u003e), whereby nucleic acid degradation occurs during the embedding process itself. We reasoned that this effect is not caused by paraffin \u003cem\u003eper se\u003c/em\u003e, but by oxygen dissolved within molten wax, which promotes oxidative DNA damage under the elevated temperatures required for embedding\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo counteract this oxidative stress, we developed an antioxidant-supplemented paraffin containing a defined mixture of Irganox-type antioxidants. Initial experiments in murine tissues demonstrated a significant improvement in DNA integrity relative to standard paraffin. These findings were subsequently extended to human tumors processed using four fixation and embedding workflows (F, FAO, G, GAO), revealing that the combined use of GAF fixation and antioxidant-supplemented paraffin consistently yielded the highest DNA integrity, reduced fragmentation, and fewer fixation-related mutational artifacts. Across sequencing platforms, DNA fragment length\u0026mdash;rather than bulk yield\u0026mdash;emerged as the primary determinant of analytical performance. This is consistent with reports showing that FFPE-induced fragmentation directly affects library complexity and coverage uniformity\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In both targeted sequencing and shallow WGS (CUTseq) assays, antioxidant-based workflows produced longer insert sizes and more homogeneous coverage, potentially improving sensitivity and reproducibility\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eVariant concordance analysis using the Jaccard Index confirmed patient-specific genotype as the dominant clustering factor but revealed reduced concordance exclusively in standard formalin/standard paraffin samples, consistent with FFPE-induced stochastic artifacts\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMutational signature analysis further distinguished biological signal from technical noise: MSI-associated signatures were preserved across workflows, whereas the FFPE-associated SBS1 signature was significantly enriched in formalin/standard paraffin samples\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e and markedly reduced by antioxidant-supplemented embedding.\u003c/p\u003e \u003cp\u003eWhole-exome sequencing showed a strong polarization of QC metrics, with GAO samples displaying superior on-target capture and coverage uniformity despite comparable throughput and other parameters critical for variant sensitivity in FFPE-based WES\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In contrast, shallow whole-genome sequencing for copy-number inference remained robust across workflows, consistent with the relative tolerance of CN analysis to moderate fragmentation\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFinally, long-read Oxford Nanopore sequencing provided a direct functional readout of high-molecular-weight DNA preservation. While total read counts were similar, GAO samples yielded significantly higher total bases and N50 values. This is particularly relevant because ONT performance depends primarily on read length rather than read number\u003csup\u003e\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecent Nanopore studies on FFPE tumors report median N50 values of ~\u0026thinsp;500\u0026ndash;600 bp\u003csup\u003e37\u003c/sup\u003e, underscoring how antioxidant-supplemented paraffin markedly exceeds conventional FFPE performance and expands the applicability of archival pathology material to long-read genomics.\u003c/p\u003e \u003cp\u003eTaken together, the results of the study show that the combination of GAF fixation with AO paraffin (GAO workflow) yielded the highest level of DNA preservation observed to date in paraffin-embedded tissues. Of interest, antioxidant supplementation also partially mitigated DNA damage in formalin-fixed tissues, indicating that formaldehyde-induced lesions arise through a multistep damage process that continues during tissue processing and can be amplified by oxidative conditions\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, this study identifies paraffin embedding as a previously underappreciated contributor to DNA degradation in histological specimens and shows that this damage can be mitigated by supplementing paraffin with lipophilic antioxidants. This simple modification preserves long DNA fragments and improves the performance of multiple sequencing applications in both formalin- and glyoxal-fixed tissues. Although validation in independent laboratories and larger cohorts will be required, particularly to assess low-frequency variant detection beyond depth-related limitations, antioxidant-supplemented paraffin represents a scalable strategy to potentially enable broad genomic utility of archival pathology specimens.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eParticipant consent Written informed consent was obtained from all participants prior to inclusion in the study, in accordance with the Declaration of Helsinki and as approved by the Institutional Ethics Review Board of the Città della Salute e della Scienza Hospital of Turin (Protocol No. 0028088; Document File No. 28/2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCM discloses support for the research of this work from:\u0026nbsp;FPRC 5 per mille MUR 2021\u0026nbsp;[CHI-RO],\u0026nbsp;FONDAZIONE AIRC under IG 2025 [ID. 32346 project],\u0026nbsp;FINPIEMONTE [P.R.F.E.S.R. 2021/27]. BB and GB disclose support from\u0026nbsp;FINPIEMONTE [Piedmont Regional Program\u0026nbsp;F.E.S.R. 2021/27] and MUR, Eurostars Project 2024,\u0026nbsp;SCRATCH (Eurostars 4508).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest:\u003c/strong\u003e\u0026nbsp; PD and CMu are employees of ADDAX Biosciences S.r.l.. BB is co-founder, and GB serves as CEO of ADDAX Biosciences S.r.l.. ADDAX Biosciences S.r.l. developed and patented GAF and AO Paraffin (\u0026ldquo;genoWax\u0026rdquo;) and provided them for the study. CM reports personal fees from an advisory board role for Roche, Illumina, AstraZeneca, Daiichi Sankyo outside the scope of the present work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e GB conceived the study and highlighting the PE Paradox and planning the use of antioxidants in preventing DNA fragmentation. EB designed the experiments, performed most of the experimental work, coordinated the study and wrote the manuscript. SEB performed the bioinformatic and computational analyses. AC and RG carried out wet-lab experiments. M.C. provided murine tissues. PD and CMu were responsible for the management and processing of human and murine samples. IC, FDG and AB performed sample collection. BB critically revised the manuscript. CM provided the financial support needed for the study, critically supported \u0026nbsp;experimental planning and data analysis, and reviewed the first draft of the manuscript. All authors approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e: all data are provided as supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability:\u003c/strong\u003e all codes are previously developed and cited in the text.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cstrong\u003ePre-clinical models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCultured human cell lines (A549, RRID:CVCL_0023, from the American Type Culture Collection and MCF-7, RRID:CVCL_0031, from NCI-60) and tissues from Balb/c mice were used for the experimental setting of the procedure. Studies on animal tissues were conducted in accordance with the national guidelines and regulations and were approved by the Italian Health Ministry (Authorization N. CC652.N.YOS).\u0026nbsp;Briefly, cells and mouse tissues (spleen, kidney, liver and HER2-overexpressing mammary tumors from BALB-neuT female mice) were fixed with either Neutral Buffered Formalin (NBF, Diapath, Martinengo (BG), Italy) or Glyoxal Acid Free (GAF) (a 2% solution of Glyoxal deprived of acids in 0.1 M phosphate buffer, pH 7.4) (ADDAX Biosciences, Turin, Italy). Following fixation for 24 h at room temperature, tissues were processed for histological analysis by dehydration, clearing and paraffin embedding using two automated tissue processors. Both systems employed standard reagents for dehydration and clearing. In one processor (Leica ASP 300, Leica Microsystems, Wetzlar, Germany), tissues were embedded using standard paraffin wax (BIO-Optica, 20134 Milan, Italy) with a melting point of 56–58°C. In the second processor (Histo PRO 300, Histoline, 20090 Pantigliate, Italy), the same paraffin wax was used but supplemented with antioxidants (AO paraffin; see below). Paraffin blocks in cassettes were then prepared using alternatively standard paraffin or AO-Paraffin. Anatomical sites of origin\u0026nbsp;were reported in\u0026nbsp;Supplementary Table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSampling was performed from 16 cases of human tumors large enough to allow multiple sampling (8 colorectal cancers, 3 renal cancers, 2 liver tumors, 1 thymoma, 1 gastric cancer and 1 ovarian cancer, Supplementary Table 4).\u0026nbsp;The study and the related project were approved by the\u0026nbsp;Board responsible for “Biobanking and use of human tissues for experimental studies” of the Department of Medical Sciences, University of Turin\u0026nbsp;and by the Institutional Ethics Review Board of the Città della Salute e della Scienza Hospital of Turin (Protocol No. 0028088; Document File No. 28/2024).\u0026nbsp;Parallel slices were fixed, two in NBF and two in GAF fixative. Following fixation at room temperature for 24 hours, NBF and the GAF-fixed tissue blocks were processed for paraffin embedding, either in the standard paraffin wax or in the AO Paraffin (see above). Overall, for each case four fixation/embedding protocols were tested: one tissue block (F) was fixed in NBF and embedded in standard paraffin; one (FAO) was fixed in NBF and embedded in paraffin supplemented with Antioxidants; one (G) was fixed in GAF and embedded in standard paraffin; one (GAO) was fixed in GAF and embedded in paraffin supplemented with Antioxidants. Analyses were therefore conducted in parallel on 64 tissue blocks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA Extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA from fixed-only cells was extracted using the DNeasy Blood \u0026amp; Tissue Kit (QIAGEN, Hilden, Germany). For fixed and embedded tissues, 3-μm-thick sections were cut from paraffin blocks and stained for Hematoxylin-Eosin and immunohistochemistry (IHC), additional 5-μm-thick sections were used for nucleic acid (NA) extraction.DNA extraction was performed following histological assessment of cellular content on hematoxylin and eosin (H\u0026amp;E)–stained sections using the QIAamp DNA FFPE Tissue Kit (QIAGEN, Hilden, Germany). Unless otherwise specified, samples were processed without the 90°C incubation step typically used for formalin crosslink reversal. This high-temperature incubation was included only in dedicated experiments designed to assess the impact of heat-induced decrosslinking on DNA fragmentation.\u0026nbsp;Nucleic acids were quantified using the Qubit fluorometer (Qubit, ThermoFisher Scientific, Waltham, MA, USA) and assessed for integrity with the Agilent 4150 TapeStation System (Agilent Technologies, Santa Clara, CA, US), returning the\u0026nbsp;DNA Integrity Number (DIN).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTargeted Next Generation Sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA purified from the 64 fixed and embedded human cancer tissues was subjected to targeted deep sequencing using the TruSight Oncology 500 panel (TSO500; Illumina, San Diego, CA, USA), which covers 523 cancer-related genes, spanning approximately 1.2 Mb of coding regions (1.94 Mb total genomic coverage). Library preparation was performed according to the manufacturer’s instructions and sequencing was carried out on an Illumina NextSeq 550 platform.\u003c/p\u003e\n\u003cp\u003eRaw sequencing data were processed using the DRAGEN on-site analysis pipeline (v4.0; Illumina) in tumor-only mode, aligned to the human reference genome GRCh37 (hg19). Variants with a variant allele frequency (VAF) \u0026gt;5% were retained and annotated using InterVar, as previously described. Putative germline variants were bioinformatically flagged by querying population databases including gnomAD Exomes, gnomAD Genomes, and the 1000 Genomes Project, applying a minimum alternative allele count of 50 and a population allele frequency threshold of ≥0.01. Remaining variants were annotated as putative somatic. TMB and MSI were calculated as previously reported\u003csup\u003e38\u003c/sup\u003e. Single base substitution (SBS) mutational signatures were inferred from targeted sequencing data generated with the TruSight Oncology 500 panel for each single fixation type. Only putative somatic variants with a variant allele frequency (VAF) \u0026gt;5% were considered for downstream analyses in the .vcf file. Somatic variants were used as input for mutational signature assignment using SigProfilerAssignment, applying reference signature sets from COSMIC Mutational Signatures v3.5 (Human Cancer) and COSMIC Experimental Signatures v1.0. Signature attribution was performed using default parameters\u003csup\u003e39\u003c/sup\u003e, and the relative contribution of each SBS signature was estimated for individual samples as well as for aggregated cohorts stratified by fixation and embedding workflow. Jaccard Index was calculated as previously reported\u003csup\u003e9\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShallow whole-genome sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShallow whole-genome sequencing (sWGS) was performed on all the 64 human cancer samples using the CUTseq protocol, as previously described and validated\u003csup\u003e40-42\u003c/sup\u003e. Libraries were prepared following the published procedure and sequenced on an Illumina NovaSeq 6000 system using a high-output 75 bp single-end configuration.\u003c/p\u003e\n\u003cp\u003eSequencing data were processed using a custom bioinformatic pipeline. Read counts were computed in fixed genomic bins of 50, 100, 250 and 500 kb, and genome-wide copy-number profiles were generated by applying circular binary segmentation to log\u003csub\u003e2\u003c/sub\u003e-transformed read counts using the DNAcopy R package (v1.74.1). Pearson correlation values were calculated for each sample within the four \u0026nbsp;different fixation protocols.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole Exome Sequencing\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWES libraries were prepared using the Agilent SureSelect Clinical Research Exome V4 (CRE V4) (Agilent, Santa Clara, CA, USA) and sequenced on an Illumina NovaSeq platform (Illumina, San Diego, CA, USA) producing 2×150bp paired-end reads. Raw sequencing data were then aligned to human reference genome hg19 using the Burrows-Wheeler Aligner (v.0.7.17), followed by BAM files sorting and indexing via SAMtools. We used Picard tools and Genome Analysis Toolkit (GATK v4.5) for duplicate marking, base quality score recalibration and coverage metrics statistics. Somatic short variant calling was performed with Mutect2 in tumor-only mode.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVariants were annotated with ANNOVAR/InterVar against the hg19 reference genome. Germline-like variants were excluded based on population allele frequencies (gnomAD, 1000 Genomes, ESP6500 ≥0.001), ClinVar benign annotations, or variant allele fraction (VAF) \u0026gt;0.9. Somatic-like variants were defined as rare or absent from population databases (\u0026lt;1×10⁻⁴), supported by high sequencing depth (DP ≥40, AD_ALT ≥5), and restricted to single-nucleotide variants. AF thresholds were set to 0.15–0.80. Variants not meeting these criteria were classified as germline proxy. For mutational signature analysis, only high-confidence somatic SNVs located in coding or splicing regions were retained. Minimal VCF files containing CHROM, POS, REF, and ALT were generated and analyzed using MutationalPatterns to derive 96-channel trinucleotide profiles\u003csup\u003e39\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOxford Nanopore Technology (ONT) sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe applied ONT, long read sequencing to 10 F- and 10 GAO-fixed tumors. 500 ng from each sample were used as input DNA to prepare libraries by means of the Native Barcoding Kit 24 V14 (Oxford Nanopore Technologies plc, UK) and then sequenced with the WGS \u0026nbsp;Ligation Sequencing Kit DNA V14 (Oxford Nanopore Technologies plc, UK) following the manufacturer's instructions. We sequenced the five libraries/flow cell on the PromethION P24 instrument. Demultiplexed FASTQ files were aligned to the human reference genome hg19 and the resulting .bam files analysed for the sequencing metrics using the NanoPlot tool (\u003ca href=\"https://github.com/wdecoster/NanoPlot\"\u003egithub.com/wdecoster/NanoPlot\u003c/a\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistics were performed with R software v4.03. Differences in distributions were analysed with a paired t-test, and contingency was assessed using\u0026nbsp;by Fisher exact test or chi-square test.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntioxidants for AO protocols\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of different liposoluble Anti-Oxidants of the Irganox type (Irganox 1010 (Pentaerythritol tetrakis(3,5-di-\u003cem\u003etert\u003c/em\u003e-butyl-4-hydroxyhydrocinnamate)), Irganox 1076 (Octadecyl 3-(3,5-di-\u003cem\u003etert\u003c/em\u003e-butyl-4-hydroxyphenyl)propionate), all from Sigma-Aldrich (Milan, Italy); and Irganox 1520 (2-Methyl-4,6-bis(octylsulfanylmethyl)phenol) from BASF Italia, (Cesano Maderno, MB, Italy) was dissolved in standard paraffin wax for histology. The deleterious effect of minor impurities present in standard paraffin on the performance of WGS sequencing (see below) could be prevented by the adoption of a highly purified paraffin wax. The resulting reagent, genoWax®, was prepared and supplied by Addax Biosciences srl, (Turin, Italy).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrior to the study, we examined the effect of adding a small amount of liposoluble antioxidants (below 0.5% w/v) to standard paraffin. Tests conducted on the technical suitability of AO paraffin (processing, sectioning), and focused on the structural features of embedded tissues (in H\u0026amp;E-stained sections) showed no appreciable differences, assuring that the antioxidant-supplemented paraffin could be safely adopted as an histological embedding medium.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003evan der Lem T, de Bakker M, Keuck G, Richardson MK (2021) Wilhelm His Sr. and the development of paraffin embedding. 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Nat Commun 12:3903. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41467-021-24078-9\u003c/span\u003e\u003cspan address=\"10.1038/s41467-021-24078-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8787286/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8787286/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDNA analysis from formalin-fixed paraffin-embedded (FFPE) tissues is frequently compromised by fragmentation and fixation-induced artifacts, limiting advanced genomic applications. While fixation chemistry has been extensively studied, the contribution of paraffin embedding to DNA damage remains poorly defined.\u003c/p\u003e \u003cp\u003eHere, starting from a direct experimental observation of embedding-associated DNA degradation, we identify paraffin embedding as an unrecognized source of oxidative DNA damage that can be mitigated by supplementing paraffin with lipophilic antioxidants. We analysed 190 samples spanning pre-clinical and clinical settings, including 126 murine specimens and 64 human tumors processed using four fixation and embedding workflows. Using more than 180 sequencing assays \u0026mdash; including targeted sequencing, whole-exome sequencing, shallow whole-genome sequencing, and long-read Oxford Nanopore whole-genome sequencing \u0026mdash; we show that antioxidant-supplemented paraffin consistently preserves DNA integrity, increasing DNA integrity number (DIN) values and enriching for DNA fragments exceeding 10 kb in length.\u003c/p\u003e \u003cp\u003eIn human tumors, antioxidant-supplemented paraffin enabled robust performance across all tested genomic applications, reducing fixation-related artifacts and improving compatibility with both short- and long-read sequencing. Collectively, these findings identify paraffin embedding as a key pre-analytical determinant of molecular preservation and provide a simple, scalable strategy for enabling genomic analysis from archival pathology specimens.\u003c/p\u003e","manuscriptTitle":"Innovative paraffin embedding to unlock archival blocks for highly demanding genomic analyses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-13 12:49:25","doi":"10.21203/rs.3.rs-8787286/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"35d58a72-4958-47f5-808c-0806c0435bf9","owner":[],"postedDate":"February 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":62404906,"name":"Health sciences/Molecular medicine"},{"id":62404907,"name":"Health sciences/Oncology/Cancer/Tumour biomarkers"}],"tags":[],"updatedAt":"2026-04-02T03:20:41+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-13 12:49:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8787286","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8787286","identity":"rs-8787286","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}