João Carlos Degraf Muzzi

Background/Objectives: Adrenocortical carcinoma (ACC) is a rare malignancy in which overexpression of steroidogenic factor-1 (SF-1/NR5A1) and aberrant activation of canonical Wnt/β-catenin signaling are important oncogenic events. However, the extent to which these regulatory axes converge or interact at the transcriptional level remains unclear. We investigated their relationship using bulk transcriptomic and regulatory-network approaches. Methods: Regulatory network inference using RTN/ARACNe was applied to the TCGA-ACC cohort. NR5A1 and β-catenin-associated TCF/LEF regulon activities were evaluated in perturbation datasets and tested for associations with CpG island methylator phenotype (CIMP), overall survival, and gene-level interaction effects. Candidate modulators of NR5A1 activity were assessed using the MINDy algorithm. The effects of cBAF inhibition on NR5A1 regulon activity were also evaluated in H295R and CU-ACC1 cells. Results: NR5A1 knockdown repressed steroidogenic pathways, whereas β-catenin knockdown predominantly suppressed Wnt/β-catenin signaling. In TCGA-ACC, NR5A1 regulon activity was associated with CIMP-high status and overall survival. In contrast, β-catenin-related regulons were not significantly associated with CIMP-high status after adjustment for NR5A1 activity, and no significant multiplicative interaction effects were identified. Fewer than 3% of protein-coding genes showed improved fit in models including NR5A1 × TCF/LEF interaction terms, without enrichment for steroidogenic or Wnt/β-catenin-related pathways. CTNNB1 was identified as a statistically significant but low-ranking positive modulator of NR5A1 activity, whereas CTNNBIP1 was a top-decile negative modulator. cBAF inhibition was associated with NR5A1 regulon repression in both cell models. Conclusions: These findings indicate limited detectable global transcriptional convergence between NR5A1 and canonical β-catenin-related regulons in the evaluated bulk transcriptomic frameworks. Potential relationships between these pathways may depend on more localized, chromatin-dependent, protein-level, or context-specific regulatory mechanisms. NR5A1 regulon activity showed more consistent associations with CIMP-high status and clinical outcome than the β-catenin/TCF-LEF regulons in the evaluated TCGA-ACC models. The modulation patterns associated with CTNNB1 and CTNNBIP1, together with the repression of NR5A1 regulon activity following cBAF inhibition, raise the possibility that NR5A1 acts as a context-dependent regulatory node connecting oncogenic signaling and chromatin-remodeling mechanisms in ACC. These findings support further investigation into the role of chromatin-remodeling complexes in sustaining NR5A1-driven transcriptional programs in ACC.

Neuroblastoma is the most common extracranial solid tumor in children, presenting remarkable clinical heterogeneity with survival outcomes ranging from spontaneous regression to aggressive progression. MYCN oncogene amplification and age at diagnosis are established prog-nostic factors that are typically treated as independent covariates in risk stratification, yet their joint influence on the tumor immune microenvironment remains poorly understood. Here we show that stratifying patients by both variables simultaneously reveals six reproducible immune subtypes with distinct transcriptional programs and prognostic significance. Consensus clustering of immunomodulatory gene expression profiles from 149 patients in the TARGET-NBL cohort identified subtypes whose survival trajectories differ significantly within clinical strata defined by MYCN status and age at diagnosis. A linear Support Vector Machine classifier trained on these subtypes, using immunomodulatory gene expression combined with MYCN amplification status and age at diagnosis as predictive features, achieved 97.2% accuracy and a Cohen’s Kappa of 0.963 under 10-fold cross-validation, and generalized to an independent cohort of 493 patients (GSE62564). Kaplan-Meier analysis revealed significant survival differences across subtypes in both cohorts (TARGET-NBL: log-rank p = 0.0018; GSE62564: log-rank p < 0.0001). Single-sample gene set enrichment analysis identified differential activation of proliferative and immune response pathways across subtypes, consistent between both cohorts. These findings suggest that integrating MYCN amplification status and age at diagnosis as joint determinants of immune organization may reveal prognostic heterogeneity that is not fully captured when these factors are considered independently.

Supplementary Figure 1. Posterior distribution of the Time to the Most Recent Common Ancestor (TMRCA), in years, derived from 1,000,000 Monte Carlo simulations assuming 2, 4, or 6 initial founders.

Background:The TP53 p.R337H variant is a well-characterized founder mutation with an unusually high prevalence in Brazil, particularly in the South and Southeast regions, where it affects approximately one in 300 individuals. It is recurrently associated with pediatric adrenocortical tumors and malignancies within the Li–Fraumeni syndrome spectrum but exhibits markedly incomplete penetrance, suggesting the influence of additional genetic and/or environmental modifiers. Although TP53 p.R337H occurs at low frequencies in the Iberian Peninsula, its origin, timing, and dissemination pattern within Brazil have remained unresolved.Methods:We integrated population–genetic inference with historical demographic modeling to reconstruct the introduction and expansion of the TP53 p.R337H in Brazil.Results:Our analyses support a founder effect arising from a single founding event of European origin during the early Portuguese colonization period. The subsequent geographic dissemination of the variant parallels historical population growth, particularly in southern Brazil. The striking regional enrichment can be explained without invoking multiple introduction events or complex migratory scenarios.Conclusions:These findings clarify the evolutionary and demographic history of TP53 p.R337H in Brazil and underscore its founder effect origin during European colonization.Impact:This study highlights the value of integrating population genetics with historical data to elucidate the spread of medically relevant founder variants. Understanding the dissemination of TP53 p.R337H will enhance public health planning and genetic counseling strategies in Brazil.See related In the Spotlight, p. 187

Background: Adrenocortical carcinoma (ACC) is a rare, aggressive malignancy where endogenous steroid excess may foster immune evasion. However, whether this hormonal axis directly modulates the antigen presentation machinery remains unclear. Methods: We applied an immunoinformatics approach to the TCGA-ACC cohort (n = 79) to investigate relationships among steroid phenotype, HLA expression, tumor microenvironment (TME), and patient outcome. Key findings were assessed in an independent validation cohort (ENSAT-ACC, n = 44) using C1A/C1B molecular subtypes corresponding to the steroid phenotypes. Results: Stratification by steroid phenotype revealed two distinct immunological profiles. The high steroid production (HSP) phenotype was associated with suppressed HLA expression and a lymphocyte-depleted “cold” TME. In contrast, the low steroid production (LSP) phenotype displayed elevated HLA expression, enriched T-cell infiltration, and upregulation of immune checkpoints (e.g., PDCD1, CTLA4), consistent with an inflamed but exhausted TME. The core signature of HLA downregulation in the HSP-like phenotype (C1A) and the significant survival advantage of the LSP-like phenotype (C1B) were confirmed in the validation cohort, demonstrating biological robustness despite platform and sample size differences. Conclusions: These findings identify the steroid phenotype as a critical regulator of immune escape in ACC. Our results support incorporating this stratification as a biomarker for patient selection, identifying LSP tumors as the subgroup most likely to benefit from immune checkpoint blockade due to their “hot” yet exhausted microenvironment.

The incidence of colorectal cancer (CRC) and the associated mortality in CRC patients have been rising in recent years. Quiescin sulfhydryl oxidase 1 (QSOX1), a secreted disulfide catalyst essential for extracellular matrix (ECM) assembly, is upregulated in several tumors (e.g. pancreatic, breast, and lung cancer), often correlating with aggressive tumor phenotypes and worse prognosis. In contrast, colorectal and hepatocellular carcinoma specimens show significant downregulation of QSOX1 compared to normal or adjacent tissue. Recognizing cancer as a heterocellular tissue where stromal cell types are crucial to tumor behavior, we evaluated by immunohistochemistry stromal and epithelial QSOX1 expression in 140 CRC cases (mean age: 64 years, 56% female, 83% without neoadjuvant therapy) and 10 normal colon samples. We found that stromal QSOX1 expression is significantly reduced in CRC compared to normal colon tissue (p < 0.0001). The stromal, rather than the epithelial, compartment determines total QSOX1 levels in tumor samples. Stromal QSOX1 demonstrates a negative relation with tumor size (ß = -0.04, p < 0.05), but not with other histopathological characteristics. This finding suggests that stromal QSOX1 loss may play a key role in early tumor cell proliferation rather than in clinical progression. Importantly, stromal QSOX1 expression shows excellent discriminatory power between tumor and non-tumor tissues, with an AUC of 0.98 in ROC analysis. Altogether, QSOX1 downregulation is a defining feature of tumor-associated stroma in CRC, likely affecting ECM integrity and modulating epithelial-stromal crosstalk. Thus, further characterization of stromal molecular signatures may identify novel biomarkers and therapeutic target strategies in CRC.

Leprosy remains a significant global health concern, with over 200,000 new cases reported annually. Approximately 10% of patients already have grade 2 disabilities at diagnosis, highlighting the need for better early detection tools of the infection caused by Mycobacterium leprae . To address this, a lateral flow assay (LFA) is developed as a proof of concept for innovative leprosy diagnostics. The assay utilizes gold nanoparticles decorated with keyhole limpet hemocyanin bound to synthetic epitopes of M. leprae proteins. When leprosy antibodies are present in the sample, a stained dot appears as a result of the capture of antigen–antibody complex by an anti‐human antibody adsorbed to the strip. After being evaluated with clinical samples, the LFA demonstrates a sensitivity and specificity of 91.67 and 95.45%, respectively, even when samples with low antibody titers are assayed. Its simplicity, user‐friendliness, and low cost (US$0.10 per unit) associated with its rapid results render it a promising tool for implementation in public health programs, particularly in areas with limited resources. Given its performance, this assay can be a valuable asset for the diagnostic challenges posed by leprosy, contributing to its control and elimination efforts.

Background: The TP53 p.R337H variant is a well-characterized founder mutation with an unusually high prevalence in Brazil, particularly in the South and Southeast regions, where it affects approximately one in 300 individuals. It is recurrently associated with pediatric adrenocortical tumors and malignancies within the Li–Fraumeni syndrome spectrum but exhibits markedly incomplete penetrance, suggesting the influence of additional genetic and/or environmental modifiers. Although TP53 p.R337H occurs at low frequencies in the Iberian Peninsula, its origin, timing, and dissemination pattern within Brazil have remained unresolved. Methods: We integrated population–genetic inference with historical demographic modeling to reconstruct the introduction and expansion of the TP53 p.R337H in Brazil. Results: Our analyses support a founder effect arising from a single founding event of European origin during the early Portuguese colonization period. The subsequent geographic dissemination of the variant parallels historical population growth, particularly in southern Brazil. The striking regional enrichment can be explained without invoking multiple introduction events or complex migratory scenarios. Conclusions: These findings clarify the evolutionary and demographic history of TP53 p.R337H in Brazil and underscore its founder effect origin during European colonization. Impact: This study highlights the value of integrating population genetics with historical data to elucidate the spread of medically relevant founder variants. Understanding the dissemination of TP53 p.R337H will enhance public health planning and genetic counseling strategies in Brazil. See related In the Spotlight, p. 187

Steroidogenic factor-1 (SF-1, also termed Ad4BP; NR5A1 in the official nomenclature) is a nuclear receptor transcription factor that plays a crucial role in the regulation of adrenal and gonadal development, function and maintenance. In addition to its classical role in regulating the expression of P450 steroid hydroxylases and other steroidogenic genes, involvement in other key processes such as cell survival/proliferation and cytoskeleton dynamics have also been highlighted for SF-1. SF-1 has a restricted pattern of expression, being expressed along the hypothalamic-pituitary axis and in steroidogenic organs since the time of their establishment. Reduced SF-1 expression affects proper gonadal and adrenal organogenesis and function. On the other hand, SF-1 overexpression is found in adrenocortical carcinoma and represents a prognostic marker for patients’ survival. This review is focused on the current knowledge about SF-1 and the crucial importance of its dosage for adrenal gland development and function, from its involvement in adrenal cortex formation to tumorigenesis. Overall, data converge towards SF-1 being a key player in the complex network of transcriptional regulation within the adrenal gland in a dosage-dependent manner.