NR1H3 (LXRα) is associated with pro-inflammatory macrophages, predicts survival and suggests potential therapeutic rationales in diffuse large b-cell lymphoma.

The role of macrophages (Mo) and their prognostic impact in diffuse large B-cell lymphomas (DLBCL) remain controversial. By regulating the lipid metabolism, Liver-X-Receptors (LXRs) control Mo polarization/inflammatory response, and their pharmacological modulation is under clinical investigation to treat human cancers, including lymphomas. Herein, we surveyed the role of LXRs in DLBCL for prognostic purposes. Comparing bulk tumors with purified malignant and normal B-cells, we found an intriguing association of NR1H3, encoding for the LXR-α isoform, with the tumor microenvironment (TME). CIBERSORTx-based purification on large DLBCL datasets revealed a high expression of the receptor transcript in M1-like pro-inflammatory Mo. By determining an expression cut-off of NR1H3, we used digital measurement to validate its prognostic capacity on two large independent on-trial and real-world cohorts. Independently of classical prognosticators, NR1H3high patients displayed longer survival compared with NR1H3low cases and a high-resolution Mo GEP dissection suggested a remarkable transcriptional divergence between subgroups. Overall, our findings indicate NR1H3 as a Mo-related biomarker identifying patients at higher risk and prompt future preclinical studies investigating its mouldability for therapeutic purposes.

Ovarian Stem Cells (OSCs) from the Cryopreserved Ovarian Cortex: A Potential for Neo-Oogenesis in Women with Cancer-Treatment Related Infertility: A Case Report and a Review of Literature.

Cancer treatment related infertility (CTRI) affects more than one third of young women undergoing anti-cancer protocols, inducing a premature exhaustion of the ovarian reserve. In addition to ovarian suppression by GnRHa, oocyte and cortex cryopreservation has gained interest in patients with estrogen-sensitive tumors for whom the hormonal burst to prompt the multiple follicular growth could provide a further pro-life tumor pulsing. On the other hand, cortex reimplantation implies a few drawbacks due to the unknown consistency of the follicles to be reimplanted or the risk of reintroducing malignant cells. The capability of ovarian stem cells (OCSs) from fresh ovarian cortex fragments to differentiate in vitro to mature oocytes provides a tool to overcome these drawbacks. In fact, since ovarian cortex sampling and cryopreservation is practicable before gonadotoxic treatments, the recruitment of OSCs from defrosted fragments could provide a novel opportunity to verify their suitability to be expanded in vitro as oocyte like cells (OLCs). Here, we describe in very preliminary experiments the consistency of an OSC population from a single cryopreserved ovarian cortex after thawing as well as both their viability and their suitability to be further explored in their property to differentiate in OLCs, thus reinforcing interest in stemness studies in the treatment of female CTRI.

Cryopreservation of adipose tissue with and without cryoprotective agent addition for breast lipofilling: A cytological and histological study.

In the second reconstructive phase of the breast after mastectomy, lipofilling is often necessary. Currently, lipofilling occurs immediately after autologous adipose tissue harvesting procedure, but most of the patients, usually, require multiple sessions to obtain a satisfactory result. Therefore, the need of repeated surgical harvesting outputs implies high risk of patients' morbidity and discomfort as well as increasing medical time and costs. The aim of our pilot study was to find out a feasible method to cryopreserve adipose tissue, in order to avoid reiterated liposuctions. Lipoaspirates samples have been harvested from 10 women and preserved by three methods: (1) the first one, using 10% Me2SO and 20% human albumin from human plasma as cryoprotective agents; (2) the second one, adding 5% Me2SO as cryoprotective agent; 3) the last one, without any cryoprotective agent. Fresh and cryopreserved fat samples, obtained through the aforementioned processes, have been analyzed ex vivo. The efficiency of the cryopreservation methods used was determined by adipocyte viability and the expression of adipocytes surface markers. Lipoaspirates stored at -196 °C for 3 months, after thawing, retained comparable adipocyte viability and histology to fresh tissue and no significant differences were found between the three methods used. Although the current results, differences between the methodologies in terms of viability may not become evident until breast lipofilling using frozen-thawed cryopreserved tissue.

uPAR+ extracellular vesicles: a robust biomarker of resistance to checkpoint inhibitor immunotherapy in metastatic melanoma patients.

BACKGROUND: Emerging evidence has highlighted the importance of extracellular vesicle (EV)-based biomarkers of resistance to immunotherapy with checkpoint inhibitors in metastatic melanoma. Considering the tumor-promoting implications of urokinase-type plasminogen activator receptor (uPAR) signaling, this study aimed to assess uPAR expression in the plasma-derived EVs of patients with metastatic melanoma to determine its potential correlation with clinical outcomes. METHODS: Blood samples from 71 patients with metastatic melanoma were collected before initiating immunotherapy. Tumor-derived and immune cell-derived EVs were isolated and analyzed to assess the relative percentage of uPAR+ EVs. The associations between uPAR and clinical outcomes, sex, BRAF status, baseline lactate dehydrogenase levels and number of metastatic sites were assessed. RESULTS: Responders had a significantly lower percentage of tumor-derived, dendritic cell (DC)-derived and CD8+ T cell-derived uPAR +EVs at baseline than non-responders. The Kaplan-Meier survival curves for the uPAR+EV quartiles indicated that higher levels of melanoma-derived uPAR+ EVs were strongly correlated with poorer progression-free survival (p<0.0001) and overall survival (p<0.0001). We also found a statistically significant correlation between lower levels of uPAR+ EVs from both CD8+ T cells and DCs and better survival. CONCLUSIONS: Our results indicate that higher levels of tumor-derived, DC-derived and CD8+ T cell-derived uPAR+ EVs in non-responders may represent a new biomarker of innate resistance to immunotherapy with checkpoint inhibitors. Moreover, uPAR+ EVs represent a new potential target for future therapeutic approaches.

Examining the Relationship between Circulating CD4- CD8- Double-Negative T Cells and Outcomes of Immuno-Checkpoint Inhibitor Therapy-Looking for Biomarkers and Therapeutic Targets in Metastatic Melanoma.

BACKGROUND: The role of circulating CD4-/CD8- double-negative T cells (DNTs) in the immune response to melanoma is poorly understood, as are the effects of checkpoint inhibitors on T cell subpopulations. METHODS: We performed a basal and longitudinal assessment of circulating immune cells, including DNTs, in metastatic melanoma patients treated with checkpoint blockade in a single-center cohort, and examined the correlations levels of immune cells with clinical features and therapy outcomes. RESULTS: Sixty-eight patients (48 ipilimumab, 20 PD1 inhibitors) were enrolled in the study. Our analysis indicated that better outcomes were associated with normal LDH, fewer than three metastatic sites, an ECOG performance status of 0, M1a stage, lower WBC and a higher lymphocyte count. The increase in lymphocyte count and decrease of DNTs were significantly associated with the achievement of an overall response. The median value of DNT decreased while the CD4+ and NK cells increased in patients that responded to treatment compare to those who did not respond to treatment. CONCLUSIONS: DNT cells change during treatment with checkpoint inhibitors and may be adept at sensing the immune response to melanoma. The complementary variation of DNT cells with respect to CD4+ and other immune actors may improve the reliability of lymphocyte assessment. Further investigation of DNT as a potential target in checkpoint inhibitor resistant melanoma is warranted.

Cellular analysis of bronchoalveolar lavage fluid to narrow differential diagnosis of checkpoint inhibitor-related pneumonitis in metastatic melanoma.

BACKGROUND: The diagnosis of check-point inhibitor-related pneumonitis (CIP) relies on radiological and clinical patterns which are not specific and can mimic other conditions (cancer progression, infectious diseases or interstitial pneumonitis). Cell pattern analysis of bronchoalveolar lavage (BAL) is well-known to support the diagnosis of interstitial lung disease; nevertheless, this analysis is somewhat performed and not required by immune-toxicity management guidelines for CIP. METHODS: We performed BAL analysis in 5 metastatic melanoma (MM) patients who developed CIP among 112 patients treated with checkpoint inhibitors. We also correlated the BAL features with the computed tomography (CT) scan patterns and with various peripheral blood parameters to better define the profile of this patient population. RESULTS: BAL flow cytometer and cytopathology analyses showed typical and homogeneous features with increased lymphoid population, prevalent CD8 + T cells and inversion of the CD4/CD8 ratio. Moreover, the extent of activated CD3 + HLA-DR + T cells was related to the grading of adverse events. Blood leucocytosis, hypoxemia, normal values for procalcitonin and lactate dehydrogenase were also found together with a cryptogenic organizing pneumonia-like radiologic pattern. In all our patients, CIP was associated with partial or complete response. CONCLUSIONS: Identification of a specific BAL cellular pattern allows clinicians to place this investigation in the appropriate position of CIP diagnosis and management to avoid misdiagnosis or considering this condition as progressive disease and delaying proper treatment.

Case Report: Lymphocytosis Associated With Fatal Hepatitis in a Thymoma Patient Treated With Anti-PD1: New Insight Into the Immune-Related Storm.

Recent advances in tumor immunotherapy have made it possible to efficiently unleash immune effectors, reacting against neoplastic cells. Although these approaches primarily aim to eradicate malignancy, immune-related adverse events (irAEs) often influence patients' prognosis, constituting a new spectrum of side effects. Taking into account the typical microenvironment and the intricate equilibrium between the anti-tumor response and the immune cells, the thymoma constitutes a unicum in the immune-oncology field. We report a fatal immune-mediated adverse events' storm in a thymoma patient treated with Pembrolizumab, leading to hepatotoxicity accompanied by lymphocytosis, thrombocytopenia, and thyroid dysfunction, unveiling a novel potential pathophysiological effect of immunotherapy. The clinical proficiency of the immune checkpoint inhibitors in thymoma patients warrants timely prevention and management of off-target consequences in order to optimize this promising therapeutic option. This case report describes a unique consequence of irAEs, emerging as a red flag warranting a multidisciplinary approach.

The Tumor Microenvironment of DLBCL in the Computational Era.

Among classical exemplifications of tumor microenvironment (TME) in lymphoma pathogenesis, the "effacement model" resembled by diffuse large B cell lymphoma (DLBCL) implies strong cell autonomous survival and paucity of non-malignant elements. Nonetheless, the magnitude of TME exploration is increasing as novel technologies allow the high-resolution discrimination of cellular and extra-cellular determinants at the functional, more than morphological, level. Results from genomic-scale studies and recent clinical trials revitalized the interest in this field, prompting the use of new tools to dissect DLBCL composition and reveal novel prognostic association. Here we revisited major controversies related to TME in DLBCL, focusing on the use of bioinformatics to mine transcriptomic data and provide new insights to be translated into the clinical setting.