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Background: Alveolar soft-part sarcoma (ASPS) is a rare tumor driven by the ASPSCR1-TFE3 fusion protein, with a propensity for metastasis. Prognostic factors remain poorly understood, and traditional chemotherapies are largely ineffective. Recent interest lies in immune checkpoint inhibitors (ICIs), yet predictive biomarkers for treatment response are lacking. Previous studies have shown promising results with ICIs in ASPS, indicating a need for further investigation into biomarkers associated with immune response. Objectives: To identify prognostic biomarkers in ASPS and to explore the role of immune-related markers, particularly L1CAM, in predicting patient outcomes. Design: A retrospective cohort study of 19 ASPS patients registered in the GEIS database. The study involved the collection of clinical and histopathological data, followed by an analysis of immune markers and gene expression profiles to identify potential prognostic indicators. Methods: Clinical and histopathological data were retrospectively collected from the GEIS-26 study cohort of 19 ASPS patients. Immunohistochemistry was performed to evaluate immune markers programmed death-1 ligand (PD-L1), programmed death-1, FAS, FASL, CD8, CD3, and CD4. An HTG ImmunOncology panel was conducted on formalin-fixed paraffin-embedded samples to explore gene expression. Effects of differentially expressed genes on survival were explored by Kaplan–Meier. Results: PD-L1 positivity was widely observed (63%) in tumors, and CD8+ lymphocytic infiltration was common. High CD8 density correlated with greater overall survival (OS) while not statistically significant. No associations were found for other immune markers. L1CAM was identified as differentially expressed in patients with low CD8 infiltration and correlated negatively with OS. Conclusion: High L1CAM expression correlated with poorer OS, highlighting its potential as a prognostic marker and therapeutic target in ASPS. Immunomodulatory interventions may hold promise, as evidenced by PD-L1 expression and CD8+ infiltration. Further research, including larger cohorts and international collaborations, is needed to validate these findings and explore therapeutic strategies targeting L1CAM in ASPS.

Abstract Background Approximately 15% of adult GIST patients harbor tumors that are wild-type for KIT and PDGFRα genes (KP-wtGIST). These tumors usually have SDH deficiencies, exhibit a more indolent behavior and are resistant to imatinib. Underlying oncogenic mechanisms in KP-wtGIST include overexpression of HIF1α high IGFR signaling through the MAPK pathway or BRAF activating mutation, among others. As regorafenib inhibits these signaling pathways, it was hypothesized that it could be more active as upfront therapy in advanced KP-wtGIST. Methods Adult patients with advanced KP-wtGIST after central confirmation by NGS, naïve of systemic treatment for advanced disease, were included in this international phase II trial. Eligible patients received regorafenib 160 mg per day for 21 days every 28 days. The primary endpoint was disease control rate (DCR), according to RECIST 1.1 at 12 weeks by central radiological assessment. Results From May 2016 to October 2020, 30 patients were identified as KP-wtGIST by Sanger sequencing and 16 were confirmed by central molecular screening with NGS. Finally, 15 were enrolled and received regorafenib. The study was prematurely closed due to the low accrual worsened by COVID outbreak. The DCR at 12 weeks was 86.7% by central assessment. A subset of 60% experienced some tumor shrinkage, with partial responses and stabilization observed in 13% and 87% respectively, by central assessment. SDH-deficient GIST showed better clinical outcome than other KP-wtGIST. Conclusions Regorafenib activity in KP-wtGIST compares favorably with other tyrosine kinase inhibitors, especially in the SDH-deficient GIST subset and it should be taken into consideration as upfront therapy of advanced KP-wtGIST. Trial registration ClinicalTrials.gov Identifier: NCT02638766.

Cisplatin, a chemotherapeutic drug, is known for causing gastrointestinal disorders and neuropathic pain, but its impact on visceral sensitivity is unclear. Monosodium glutamate (MSG) has been shown to improve gastrointestinal dysmotility and neuropathic pain induced by cisplatin in rats. This study aimed to determine if repeated cisplatin treatment alters visceral sensitivity and whether dietary MSG can prevent these changes. Male Wistar HAN rats were treated with saline or cisplatin (2 mg/kg/week, ip) for 5 weeks, and visceral sensitivity to intracolonic mechanical stimulation was recorded after the final cisplatin administration (week 5) and one-week post-treatment (week 6). In a second cohort, rats treated with cisplatin or saline also received MSG (4 g/L) in their drinking water, and visceral sensitivity was evaluated on week 6. Finally, the untouched distal colon was obtained from a third cohort of animals one week after treatment to assess immunocyte infiltration. Cisplatin significantly increased colonic mechanical sensitivity on week 6 but not on week 5. MSG did not prevent cisplatin-induced visceral hypersensitivity on week 6 and even exacerbated it. On week 6, compared with the control, cisplatin (with or without MSG) did not modify the colonic infiltration of eosinophils, macrophages, neutrophils, or mast cells. Although MSG seems to be useful in ameliorating some of the adverse effects of cisplatin, such as gastrointestinal motility disturbances or neuropathic pain, it does not alleviate visceral pain.

In eutherian mammals, hundreds of programmed DNA double-strand breaks (DSBs) are generated at the onset of meiosis. The DNA damage response is then triggered. Although the dynamics of this response is well studied in eutherian mammals, recent findings have revealed different patterns of DNA damage signaling and repair in marsupial mammals. To better characterize these differences, here we analyzed synapsis and the chromosomal distribution of meiotic DSBs markers in three different marsupial species (Thylamys elegans, Dromiciops gliorides, and Macropus eugenii) that represent South American and Australian Orders. Our results revealed inter-specific differences in the chromosomal distribution of DNA damage and repair proteins, which were associated with differing synapsis patterns. In the American species T. elegans and D. gliroides, chromosomal ends were conspicuously polarized in a bouquet configuration and synapsis progressed exclusively from the telomeres towards interstitial regions. This was accompanied by sparse H2AX phosphorylation, mainly accumulating at chromosomal ends. Accordingly, RAD51 and RPA were mainly localized at chromosomal ends throughout prophase I in both American marsupials, likely resulting in reduced recombination rates at interstitial positions. In sharp contrast, synapsis initiated at both interstitial and distal chromosomal regions in the Australian representative M. eugenii, the bouquet polarization was incomplete and ephemeral, γH2AX had a broad nuclear distribution, and RAD51 and RPA foci displayed an even chromosomal distribution. Given the basal evolutionary position of T. elegans, it is likely that the meiotic features reported in this species represent an ancestral pattern in marsupials and that a shift in the meiotic program occurred after the split of D. gliroides and the Australian marsupial clade. Our results open intriguing questions about the regulation and homeostasis of meiotic DSBs in marsupials. The low recombination rates observed at the interstitial chromosomal regions in American marsupials can result in the formation of large linkage groups, thus having an impact in the evolution of their genomes.

During meiotic prophase I, homologous chromosomes pair, synapse and recombine in a tightly regulated process that ensures the generation of genetically variable haploid gametes. Although the mechanisms underlying meiotic cell division have been well studied in model species, our understanding of the dynamics of meiotic prophase I in non-traditional model mammals remains in its infancy. Here, we reveal key meiotic features in previously uncharacterised marsupial species (the tammar wallaby and the fat-tailed dunnart), plus the fat-tailed mouse opossum, with a focus on sex chromosome pairing strategies, recombination and meiotic telomere homeostasis. We uncovered differences between phylogroups with important functional and evolutionary implications. First, sex chromosomes, which lack a pseudo-autosomal region in marsupials, had species specific pairing and silencing strategies, with implications for sex chromosome evolution. Second, we detected two waves of γH2AX accumulation during prophase I. The first wave was accompanied by low γH2AX levels on autosomes, which correlated with the low recombination rates that distinguish marsupials from eutherian mammals. In the second wave, γH2AX was restricted to sex chromosomes in all three species, which correlated with transcription from the X in tammar wallaby. This suggests non-canonical functions of γH2AX on meiotic sex chromosomes. Finally, we uncover evidence for telomere elongation in primary spermatocytes of the fat-tailed dunnart, a unique strategy within mammals. Our results provide new insights into meiotic progression and telomere homeostasis in marsupials, highlighting the importance of capturing the diversity of meiotic strategies within mammals.

Sex chromosomes of eutherian mammals are highly different in size and gene content, and share only a small region of homology (pseudoautosomal region, PAR). They are thought to have evolved through an addition-attrition cycle involving the addition of autosomal segments to sex chromosomes and their subsequent differentiation. The events that drive this process are difficult to investigate because sex chromosomes in almost all mammals are at a very advanced stage of differentiation. Here, we have taken advantage of a recent translocation of an autosome to both sex chromosomes in the African pygmy mouse Mus minutoides, which has restored a large segment of homology (neo-PAR). By studying meiotic sex chromosome behavior and identifying fully sex-linked genetic markers in the neo-PAR, we demonstrate that this region shows unequivocal signs of early sex-differentiation. First, synapsis and resolution of DNA damage intermediates are delayed in the neo-PAR during meiosis. Second, recombination is suppressed or largely reduced in a large portion of the neo-PAR. However, the inactivation process that characterizes sex chromosomes during meiosis does not extend to this region. Finally, the sex chromosomes show a dual mechanism of association at metaphase-I that involves the formation of a chiasma in the neo-PAR and the preservation of an ancestral achiasmate mode of association in the non-homologous segments. We show that the study of meiosis is crucial to apprehend the onset of sex chromosome differentiation, as it introduces structural and functional constrains to sex chromosome evolution. Synapsis and DNA repair dynamics are the first processes affected in the incipient differentiation of X and Y chromosomes, and they may be involved in accelerating their evolution. This provides one of the very first reports of early steps in neo-sex chromosome differentiation in mammals, and for the first time a cellular framework for the addition-attrition model of sex chromosome evolution.

Homologous recombination (HR) is the principal mechanism of DNA repair acting during meiosis and is fundamental for the segregation of chromosomes and the increase of genetic diversity. Nevertheless, non-homologous end joining (NHEJ) mechanisms can also act during meiosis, mainly in response to exogenously-induced DNA damage in late stages of first meiotic prophase. In order to better understand the relationship between these two repair pathways, we studied the response to DNA damage during male mouse meiosis after gamma radiation. We clearly discerned two types of responses immediately after treatment. From leptotene to early pachytene, exogenous damage triggered the massive presence of γH2AX throughout the nucleus, which was associated with DNA repair mediated by HR components (DMC1 and RAD51). This early pathway finished with the sequential removal of DMC1 and RAD51 and was no longer inducible at mid pachytene. However, from mid-pachytene to diplotene, γH2AX appeared as large discrete foci. This late repair pattern was mediated initially by NHEJ, involving Ku70 and XRCC4, which were constitutively present, and 53BP1, which appeared at sites of damage soon after irradiation. Nevertheless, 24 hours after irradiation, a HR pathway involving RAD51 but not DMC1 mostly replaced NHEJ. Additionally, we observed the occurrence of synaptonemal complex bridges between bivalents, most likely representing chromosome translocation events that may involve DMC1, RAD51 or 53BP1. Our results reinforce the idea that the early "meiotic" repair pathway that acts by default at the beginning of meiosis is replaced from mid-pachytene onwards by a "somatic-like" repair pattern. This shift might be important to resolve DNA damage (either endogenous or exogenous) that could not be repaired by the early meiotic mechanisms, for instance those in the sex chromosomes, which lack a homologous chromosome to repair with. This transition represents another layer of functional changes that occur in meiotic cells during mid pachytene, in addition to epigenetic reprograming, reactivation of transcription, changes in the gene expression profile and acquisition of competence to proceed to metaphase.