Peter Schirmacher

Introduction: Different molecular subtypes of hepatocellular adenoma (HCA) can occur at the same time within the same patient, defining inter‐tumour heterogeneity (ITH). We describe a series of HCA‐ITH cases, most identified in Bordeaux with additional cases contributed by other international centres. Methods: ITH‐HCA cases were subtyped by immunohistochemistry, confirmed by molecular analysis in most cases. The resulting diagnoses were HNF1A ‐HCA (H‐HCA), inflammatory HCA (IHCA), b‐catenin‐mutated HCA (b‐HCA) involving exon 3 or 7/8, b‐catenin‐mutated IHCA (b‐IHCA) involving exon 3 or 7/8, sonic hedgehog HCA (sh‐HCA) and unclassified HCA (UHCA). Results: We identified 26 patients with ITH‐HCA including 17 women; none had underlying liver disease, whereas most men did (8/9). The largest subgroup comprised 22 patients with IHCA combined with another subtype: H‐HCA (five patients); H‐HCA and b‐IHCA‐exon 3 borderline (one patient); b‐IHCA‐exon 3 (six patients); b‐HCA and b‐IHCA exon 3 (four patients); b‐IHCA‐exon7/8 (two patients); sh‐HCA (three patients); UHCA (one patient). The remaining four patients had sh‐HCA combined with UHCA (one patient) or b‐IHCA ex3 (one patient), and 2 patients with HNF1A adenomatoses, one with malignant transformation, the other associated with a b‐IHCA exon3 progressed to HCC. The largest nodule was a 13 cm IHCA. One of the sh‐HCA presented with bleeding. Conclusion: ITH‐HCA covers the whole spectrum of the HCA classification. It is rare but poses significant clinical challenges, particularly in terms of identifying those lesions at risk of malignant transformation (b‐HCA or b‐IHCA exon 3) or bleeding (sh‐HCA). Referral to a special tertiary centre is recommended.

Aims: Hybrid lesions containing distinct components of hepatocellular adenoma (HCA) and focal nodular hyperplasia (FNH) within a single nodule are exceptionally rare. This study aimed to investigate the biological nature of these unusual lesions. Methods and results: The two cases identified to date were examined by histopathological, immunohistochemical and targeted next‐generation sequencing analyses. In case 1, the hybrid tumour (6.0 cm) consisted of approximately equal proportions of HNF1α‐inactivated HCA and FNH, which were intimately admixed but remained histologically distinct. In case 2, the tumour (9.5 cm) consisted predominantly of HNF1α‐inactivated HCA but contained a small peripheral nodule of FNH, resulting in a nodule‐in‐nodule appearance. The HCA components in both cases showed loss of FABP1 expression, whereas the FNH components retained FABP1 expression and showed a map‐like pattern of glutamine synthetase expression. Next‐generation sequencing in case 1 demonstrated that the mutations were not shared between the HCA and FNH components, with HNF1A and FLT1 mutations identified only in the HCA component. In case 2, the same HNF1A splice‐site mutation was identified in both the HCA and FNH components, but was also present in the non‐neoplastic gallbladder, confirming its germline origin. The HCA component, but not the FNH component, additionally harboured somatic mutations in ATM and FAT4 . Conclusion: HCA, particularly the HNF1α‐inactivated subtype, rarely coexists with FNH. The distinct molecular and immunohistochemical profiles of the two components support a collision mechanism, indicating that the FNH component represents a separate non‐neoplastic lesion and arguing against one component arising from the other.

Background: The past decade has seen the development of epigenetic models of aging that accurately estimate chronological age and predict disease incidence and mortality. These estimates are modulated by lifestyle and environmental factors linked to carcinogenesis, but to date this has primarily been studied in blood. Methods: We examined epigenetic aging in normal colonic tissue (n = 96), adjacent mucosa (n = 245), and tumors (n = 208), using models trained on age (Horvath, Hannum, Zhang), mortality (PhenoAge, GrimAge), aging rate (DunedinPACE), cellular mitotic history (EpiTOC, epiTOC2, miAGe), and telomere length (DNAmTL). Results: The Horvath model was the most accurate estimator of chronological age in normal colonic mucosa, with high correlation (r > 0.70) between the Horvath, Hannum, Zhang, PhenoAge, and GrimAge models as well as between mitotic clocks (r > 0.94). All models showed similar performance in normal tissue and adjacent mucosa but substantially more variation in estimates in tumors. Statistically significant differences in age acceleration were present between normal and adjacent mucosa by 6 models (Hannum, Zhang, PhenoAge, EpiTOC, epiTOC2, and miAge), while tumors showed highly statistically significant differences by all models. Age acceleration differed by region of the colon, with varying patterns by model type. Physical activity (PhenoAge), smoking history (GrimAge), and alcohol consumption (Horvath, mitotic clocks) were associated with epigenetic aging in adjacent mucosa, while smoking history, smoking intensity, and alcohol consumption were associated with DNAmTL in tumors. Conclusions: Our study reveals an impact of tissue type, region, and lifestyle factors on epigenetic aging and highlights heterogeneity between models and the need for careful consideration within study design.

Supplementary Table S1. Multivariate Analysis for the Hypoxia-M and Brooks signature.

Supplementary Figure S3. (Top) Hypoxia-M V2 is associated with decreased OS in the TCGApancancer cohort (p<0.002) (Bottom) Hypoxia-M V2 is an independent prognosticator of OS (HR=1.81, p<0.03) after adjusting for demographic and clinical factors.

Supplementary Table S4. Correlations with CIBERSORT immune signature expressions in the TCGA-HNSCC cohort

Supplementary Table S6. Contingency Table of Hypoxia-M and CD8-immunohistochemistry (IHC) staining for CD8-T cells