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Supplementary Table S2-S4. Pathway enrichment analysis by DAVID and GSEA in the ileum and colon of antibiotic treated mice.

1. Supplementary Materials and Methods Table S1. List of the antibodies used for flow cytometry 2. Supplementary Figures Supplementary Fig. S1. Impact of antibiotic treatment on trastuzumab efficacy and the tumor microenvironment. Supplementary Fig. S2. Depletion of commensal microbiota by antibiotic cocktail (ABX) treatment and fecal microbiota transplantation. Supplementary Fig. S3. Impact of vancomycin on trastuzumab efficacy in Î"16HER2 transgenic FVB mice. Supplementary Fig. S4. Differentially abundant bacteria in the gut microbiota of antibiotic-treated mice. Supplementary Fig. S5. Short-chain fatty acids (SCFAs) quantification in fecal samples from antibiotic-treated mice. Supplementary Fig. S6. Analysis of intratumor and stromal cell staining in tumors of antibiotic-treated mice. Supplementary Fig. S7. Impact of antibiotic treatment on tumor immune infiltrate. Supplementary Fig. S8. Analysis of intratumor and stromal cell staining in tumors from FMT mice. Supplementary Fig. S9. Impact of vancomycin treatment on intestinal and systemic immune features. Supplementary Fig. S10. Impact of IL12p70 and CD4+ cell depletion on trastuzumab antitumor efficacy and on tumor immune infiltrate. Supplementary Fig. S11. Causal role of human commensal bacteria in immune-mediated trastuzumab antitumor efficacy. 3. Supplementary References

Emerging evidence indicates that gut microbiota affect the response to anticancer therapies by modulating the host immune system. In this study, we investigated the impact of gut microbiota on immune-mediated trastuzumab antitumor efficacy in preclinical models of HER2-positive breast cancer and in 24 patients with primary HER2-positive breast cancer undergoing trastuzumab-containing neoadjuvant treatment. In mice, the antitumor activity of trastuzumab was impaired by antibiotic administration or fecal microbiota transplantation from antibiotic-treated donors. Modulation of the intestinal microbiota was reflected in tumors by impaired recruitment of CD4+ T cells and granzyme B–positive cells after trastuzumab treatment. Antibiotics caused reductions in dendritic cell (DC) activation and the release of IL12p70 upon trastuzumab treatment, a mechanism that was necessary for trastuzumab effectiveness in our model. In patients, lower α-diversity and lower abundance of Lachnospiraceae, Turicibacteraceae, Bifidobacteriaceae, and Prevotellaceae characterized nonresponsive patients (NR) compared with those who achieved pathologic complete response (R), similar to antibiotic-treated mice. The transfer of fecal microbiota from R and NR into mice bearing HER2-positive breast cancer recapitulated the response to trastuzumab observed in patients. Fecal microbiota β-diversity segregated patients according to response and positively correlated with immune signature related to interferon (IFN) and NO2-IL12 as well as activated CD4+ T cells and activated DCs in tumors. Overall, our data reveal the direct involvement of the gut microbiota in trastuzumab efficacy, suggesting that manipulation of the gut microbiota is an optimal future strategy to achieve a therapeutic effect or to exploit its potential as a biomarker for treatment response.Significance:Evidence of gut microbiota involvement in trastuzumab efficacy represents the foundation for new therapeutic strategies aimed at manipulating commensal bacteria to improve response in trastuzumab-resistant patients.See related commentary by Sharma, p. 1937

INTRODUCTION Disorders of arousal (DOA) are parasomnias that emerge from incomplete arousal out of Non-Rem Sleep (NREM) and lead to a broad variety of emotional and motor behaviours. Increasing evidence supports the hypothesis that specific psychopathological traits contribute to the multifactorial origin of these phenomena. The aim of the current multicenter study was to compare the personality profile of children and adolescents with and without DOA using the Junior Temperament and Character Inventory (JTCI). METHODS We enrolled 36 patients with a diagnosis of DOA (mean age of 11 ± 3 years, 64% males), and 36 healthy age and gender matched control subjects (mean age of 11.2 ± 3.6, years, 67% males). Their parents completed the Paris Arousal Disorder Severity Scale (PADSS), the Sleep Disturbance Scale for Children (SDSC) and the JTCI. RESULTS Patients with DOA reached significantly higher levels compared to their control group in total PADSS (p

Myoid gonadal stromal tumor represents a rare testicular neoplasm displaying smooth muscular and gonadal stromal differentiation. This entity has very few cases reported in the literature that describe heterogeneous clinical and pathological characteristics. Bayesian statistics provides a useful framework to combine information from diverse sources. We here presented a case series-the largest so far reported-of myoid gonadal stromal tumor (4 cases) with extensive morphologic, immunohistochemical, and molecular characterization, performed a systematic review of the literature (that identified 9 papers), and used a Bayesian data analysis to understand the characteristics of this disease. Our study collectively described 16 cases. This neoplasm is mainly found in adults (mean age about 40 years) and often has a size of about 3 cm. By morphology, the tumor can infiltrate testicular tubules and is composed of spindle cells; few mitoses can be seen (usually 2/10 HPF). Neoplastic cells are diffusely positive with α-smooth muscle actin with a tram-track staining pattern. S100 protein, FOXL2, and SF1 are also characteristically positive. Moreover, this neoplasm can display epithelial differentiation, in about half of the cases. In conclusion, we foresee the use of this statistical approach in pathology: our analysis allowed a more precise description of this rare entity.

Aims: To investigate the morphological and molecular characteristics of Leydig cell tumours (LCTs) of the testis for the identification of cases that may metastasise. Methods and results: Six parameters for a predictive model of the metastatic risk were evaluated in 37 benign and 14 malignant LCTs of the testis [LCT Scaled Score (LeSS)]. The tumour size (benign LCTs, mean 13.3mm; malignant LCTs, mean 44mm) (P&nbsp

International audience; Aims Renal cell carcinomas (RCCs) represent 2-5% of kidney malignancies in children and adolescents. Appropriate diagnostic and classification are crucial for the correct management of the patients and in order to avoid inappropriate pre-operative chemotherapy, which is usually recommended if a Wilms' tumour is suspected. Methods and results A French-Italian series of 93 renal cell carcinomas collected from 1990 to 2019 in patients aged less than 18 years was reclassified according to the 2016 World Health Organization (WHO) classification and the latest literature. TFE3 and TFEB fluorescence in-situ hybridisation (FISH) analyses and a panel of immunohistochemical stains were applied. The median age at diagnosis was 11 years (range = 9 months-17 years). MiT family (MiTF) translocation RCCs accounted for 52% of the tumours, followed by papillary (20%) and unclassified RCCs (13%). Other subtypes, such as SDHB-deficient and fumarate hydratase-deficient RCCs, represented 1-3% of the cases. We also described a case of ALK-rearranged RCC with a metanephric adenoma-like morphology. Conclusion A precise histological diagnosis is mandatory, as targeted therapy could be applied for some RCC subtypes, i.e. MiTF-translocation and ALK-translocation RCC. Moreover, some RCC subtypes may be associated with a predisposition syndrome that will impact patients' and family's management and genetic counselling. A precise RCC subtype is also mandatory for the clinical management of the patients and inclusion in new prospective clinical trials.

This study aimed at unravelling whether WDLPS undergo a progressive evolution to DDLPS through analyzing the transcriptomic profile of retroperitoneal liposarcoma. In a contemporary retrospective series of primary retroperitoneal DDLPS and WDLPS (N=107), we sampled paired DD/WD/normal fat (NF) components of DDLPS and paired WD/NF components of WDLPS. RNA-Seq data was normalized using the trimmed mean of M-value (TMM) algorithm, heteroscedasticity was removed, and differential expression analysis (DEA) performed. Gene sets enrichment analysis (GSEA) evaluated enrichment in biological hallmarks and was performed to overcome FDR correction of DEA and evaluate the ensemble. FDR threshold 0.05 was considered for significance. Marked transcriptional changes exist among paired components of DDLPS (DD, WD, NF) or WDLPS (WD, NF). Although DEA did not show significant changes between WD and NF components of DDLPS and WDLPS, GSEA analysis highlighted deregulation in hallmarks. An upregulation of G2M checkpoint and mitotic spindle gene sets was observed when WD components of DDLPS was compared to WDLPS and progressively increase in the DD component. Some targetable genes from the leading edge of these pathways (PLK1, AURKA, and EZH2) as well as the primary oncogenic drivers (MDM2, CDK4, and HMGA2) of liposarcoma were functionally validated with targeted agents in cell lines of DDLPS and WDLPS, supporting findings of transcriptomic analysis. We also observed that adipogenesis, fatty acid metabolism, cholesterol homeostasis, oxidative phosphorylation, and peroxisome gene sets were down-regulated in the DD component, while glycolysis was upregulated compared to WD components. When NF were compared with their paired tumor components, G2M checkpoint and mitotic spindle gene sets did not differ between DDLPS and WDLPS, suggesting these two hallmarks as tumor-specific. NF of patients with DDLPS demonstrated higher expression of adipogenesis, and other pathways related to metabolism suggesting its metabolic activation compared to NF of patients with WDLPS. In conclusion, changes distinguishing WDLPS/DDLPS early at the WD stage and progressively increasing in the DD component of DDLPS supported the hypothesis of an orderly progression from WDLPS to DDLPS and represent a source of additional therapeutic targets. Citation Format: Sandro Pasquali, Stefano Percio, Dario Callegaro, Silvia Martini, Alessia Beretta, Alessia Bertolotti, Silvia Brich, Paola Collini, Marta Barisella, Loris De Cecco, Viviana Vallacchi, Silvia Stacchiotti, Matteo Benelli, Alessandro Gronchi, Nadia Zaffaroni. The transcriptomic profile of retroperitoneal primary well differentiated liposarcoma (WDLPS) and well differentiated (WD)/dedifferentiated (DD) components of DD liposarcoma (DDLPS) reveals the progression from WDLPS to DDLPS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2245.

Soft tissue sarcomas (STSs) are a family of rare malignant tumors encompassing more than 80 histologies. Current therapies for metastatic STS, a condition that affects roughly half of patients, have limited efficacy, making innovative therapeutic strategies urgently needed. From a molecular point of view, STSs can be classified as translocation-related and those with a heavily rearranged genotype. Although only the latter display an increased mutational burden, molecular profiles suggestive of an “immune hot” tumor microenvironment are observed across STS histologies, and response to immunotherapy has been reported in both translocation-related and genetic complex STSs. These data reinforce the notion that immunity in STSs is multifaceted and influenced by both genetic and epigenetic determinants. Cumulative evidence indicates that a fine characterization of STSs at different levels is required to identify biomarkers predictive of immunotherapy response and to discover targetable pathways to switch on the immune sensitivity of “immune cold” tumors. In this review, we will summarize recent findings on the interplay between genetic landscape, molecular profiling and immunity in STSs. Immunological and molecular features will be discussed for their prognostic value in selected STS histologies. Finally, the local and systemic immunomodulatory effects of the targeted drugs imatinib and sunitinib will be discussed.

Whereas 90% of patients with Wilms tumor (WT) reach cure, approximately half of patients developing a recurrent tumor die of the disease. Therefore, to disclose events leading to recurrence represents a clinical need. To study paired primary/recurrent tumor samples, being aware of the intra-tumoral heterogeneity, might help finding these answers. We previously suggested that mutations in SIX1 and DROSHA underlie WT recurrence. With the aim to better investigate this scenario, we collected 19 paired primary/recurrent tumors and 10 primary tumors from relapsing patients and searched for mutations in the SIX1/2 genes and microRNA processing genes (miRNAPGs). We found SIX1 mutation in one case, miRNAPGs mutations in seven cases, and the co-occurrence of SIX1 and miRNAPG mutations in one case. We could observe that, whereas in primary tumors the mutations could be heterogeneously present, in all cases they were positively selected and homogeneously present in the recurrent disease, as also indicated by a “moderate” and “almost perfect” agreement (according to the Landis and Koch classification criteria) between paired samples. Analysis of SIX1/2 genes and miRNAPGs in 50 non-relapsing WTs disclosed SIX2 mutation in one case and miRNAPGs mutations in seven. A borderline statistically significant association was observed between miRNAPGs mutations and the occurrence of relapse (p value: 0.05). These data suggest that SIX1 and miRNAPGs mutations may provide an advantage during tumor progression to recurrence and can represent oncogenic drivers in WT development.

Emerging evidence indicates that gut microbiota affect the response to anticancer therapies by modulating the host immune system. In this study, we investigated the impact of gut microbiota on immune-mediated trastuzumab antitumor efficacy in preclinical models of HER2-positive breast cancer and in 24 patients with primary HER2-positive breast cancer undergoing trastuzumab-containing neoadjuvant treatment. In mice, the antitumor activity of trastuzumab was impaired by antibiotic administration or fecal microbiota transplantation from antibiotic-treated donors. Modulation of the intestinal microbiota was reflected in tumors by impaired recruitment of CD4+ T cells and granzyme B–positive cells after trastuzumab treatment. Antibiotics caused reductions in dendritic cell (DC) activation and the release of IL12p70 upon trastuzumab treatment, a mechanism that was necessary for trastuzumab effectiveness in our model. In patients, lower α-diversity and lower abundance of Lachnospiraceae, Turicibacteraceae, Bifidobacteriaceae, and Prevotellaceae characterized nonresponsive patients (NR) compared with those who achieved pathologic complete response (R), similar to antibiotic-treated mice. The transfer of fecal microbiota from R and NR into mice bearing HER2-positive breast cancer recapitulated the response to trastuzumab observed in patients. Fecal microbiota β-diversity segregated patients according to response and positively correlated with immune signature related to interferon (IFN) and NO2-IL12 as well as activated CD4+ T cells and activated DCs in tumors. Overall, our data reveal the direct involvement of the gut microbiota in trastuzumab efficacy, suggesting that manipulation of the gut microbiota is an optimal future strategy to achieve a therapeutic effect or to exploit its potential as a biomarker for treatment response. Significance: Evidence of gut microbiota involvement in trastuzumab efficacy represents the foundation for new therapeutic strategies aimed at manipulating commensal bacteria to improve response in trastuzumab-resistant patients. See related commentary by Sharma, p. 1937

e23520 Background: Retroperitoneal DDLPS is an aggressive tumor characterized by a WD component, morphologically similar to a WDLPS, and a DD component that drives patient prognosis. We aimed at unraveling whether WDLPS and DDLPS are distinct entities or entail a progressive evolution from WDLPS to DDLPS through a transcriptomic analysis. Methods: A transcriptomic analysis was performed in a retrospective series of 107 patients with primary retroperitoneal WDLPS (N = 68, 63.5%) or DDLPS (N = 39, 36.5%) who underwent surgery (2011-15). Paired DD, WD, and normal fat (NF) components were sampled in DDLPS, while paired WD and NF components were sampled in WDLPS. RNA-Seq data were normalized according to the trimmed mean of M-value (TMM) algorithm and differential expression was evaluated with the voom method implemented into the edgeR package. Enrichment in hallmark gene sets from Molecular Signatures Database (MSigDB) was evaluated with gene sets enrichment analysis (GSEA), by using t-statistic as measure of ranking. A false discovery rate (FDR) adjusted p-value < 0.05 was considered for statistical significance. Results: Differential expression analysis revealed marked transcriptional changes within paired components of DDLPS (DD, WD, NF) and WDLPS (WD, NF). Changes of WD and NF components between WDLPS and DDLPS were not statistically significant. Gene sets were analyzed to evaluate the ensemble and overcome the FDR correction applied to single genes. Hallmarks deregulated in WD component of DDLPS compared to WDLPS were detected also in their paired DD component. Among them, G2M checkpoint and mitotic spindle were up-regulated, while adipogenesis, fatty acid metabolism, cholesterol homeostasis, oxidative phosphorylation, and peroxisome were down-regulated. These differences persisted also when NF components were compared with their paired WD component of DDLPS and WDLPS. G2M checkpoint and mitotic spindle gene sets did not differ between NF of DDLPS and NF of WDLPS, suggesting these two hallmarks as tumor-specific. Conversely, expression of adipogenesis, fatty acid metabolism, and oxidative phosphorylation was up-regulated together with other gene sets related to metabolism in NF of DDLPS. Tumor inflammation and interferon response were up-regulated in WD components compared to their paired NF components. Also, interferon response was down-regulated in WD component of DDLPS compared to WDLPS, and inflammation was down-regulated in DD component compared to both WD components. Conclusions: Transcriptomic changes that distinguished WDLPS and WD component of DDLPS increased progressively also in the paired DD component of DDLPS, supporting the hypothesis of a progression from WDLPS to DDLPS in some liposarcomas. Changes observed in NF may suggest a paracrine effect sustaining tumor dedifferentiation.

Background High-grade serous ovarian cancer (HGSOC) is a major unmet need in oncology. The remaining uncertainty on its originating tissue has hampered the discovery of molecular oncogenic pathways and the development of effective therapies. Methods We used an approach based on the retention in tumors of a DNA methylation trace (OriPrint) that distinguishes the two putative tissues of origin of HGSOC, the fimbrial (FI) and ovarian surface epithelia (OSE), to stratify HGSOC by several clustering methods, both linear and non-linear. The identified tumor subtypes (FI-like and OSE-like HGSOC) were investigated at the RNAseq level to stratify an in-house cohort of macrodissected HGSOC FFPE samples to derive overall and disease-free survival and identify specific transcriptional alterations of the two tumor subtypes, both by classical differential expression and weighted correlation network analysis. We translated our strategy to published datasets and verified the co-occurrence of previously described molecular classification of HGSOC. We performed cytokine analysis coupled to immune phenotyping to verify alterations in the immune compartment associated with HGSOC. We identified genes that are both differentially expressed and methylated in the two tumor subtypes, concentrating on PAX8 as a bona fide marker of FI-like HGSOC. Results We show that: - OriPrint is a robust DNA methylation tracer that exposes the tissue of origin of HGSOC. - The tissue of origin of HGSOC is the main determinant of DNA methylation variance in HGSOC. - The tissue of origin is a prognostic factor for HGSOC patients. - FI-like and OSE-like HGSOC are endowed with specific transcriptional alterations that impact patients’ prognosis. - OSE-like tumors present a more invasive and immunomodulatory phenotype, compatible with its worse prognostic impact. - Among genes that are differentially expressed and regulated in FI-like and OSE-like HGSOC, PAX8 is a bona fide marker of FI-like tumors. Conclusions Through an integrated approach, our work demonstrates that both FI and OSE are possible origins for human HGSOC, whose derived subtypes are both molecularly and clinically distinct. These results will help define a new roadmap towards rational, subtype-specific therapeutic inroads and improved patients’ care.