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Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors with an annual incidence of ~2 cases per million worldwide. The hereditary form is more likely to present in younger patients. To date, PPGL is considered a complex pathology that is difficult to diagnose. The present study aimed to improve the molecular diagnosis and other driver mutations related to PPGLs using TruSight One clinical exome panel (Illumina, Inc.). The clinical protocol used involved examining 28 patients with suspicion of genetic alterations as the cause of PPGLs. The variants of genes commonly associated with PPGLs ( RET , FH , VHL , SDHA , SDHB , SDHC , SDHD , NF1 , MAX , HIF2A , TMEM127 and TP53 ) were filtered across the panel. The libraries were sequenced on a MiSeq instrument (Illumina, Inc.) and the result was ≥20X coverage on 95% of the target regions in the panel, calculated by averaging the mean coverage for each exon. The results of sequencing detected 7% of pathogenic variants in the 18-40 years age subgroup and 11% in the 41-59 years age subgroup, whereas no pathogenic/likely pathogenic variants were identified in patients ≥60 years old. The identification of a germline mutation in patients with apparently sporadic PPGLs could lead to an early diagnosis of multiple or more aggressive tumors, or other neoplastic syndromes, in patients. Furthermore, this information may improve the development of targeted primary and secondary prevention programs tailored to these high-risk groups., Competing Interests: The authors declare that they have no competing interests., (Copyright: © 2024 Melli et al.)

Purpose: The National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) trial was implemented to identify actionable genetic alterations across cancer types and enroll patients accordingly onto treatment arms, irrespective of tumor histology. Using multiplex polymerase chain reaction (PCR) next-generation sequencing, NCI-MATCH genotyped 5,540 patients, discovering gene fusions in 202/5,540 tumors (3.65%). This result, substantially lower than the fusion detection prevalence of 8.5% across all patients with cancer screened at Massachusetts General Hospital's (MGH) clinical laboratories, supported reanalysis of NCI-MATCH samples identified as mutations-of-interest (MOI)-negative. The assay used by NCI-MATCH requires previous knowledge of both fusion genes, cannot detect novel fusions, and may underestimate fusion-positive patients. Anchored multiplex PCR (AMP) technology permits fusion detection with knowledge of just one gene of the fusion partners., Methods: Using AMP-based kits, we reprocessed 663 MOI-negative samples. 200 ng of RNA per sample were shipped from the Eastern Cooperative Oncology Group-American College of Radiology Imaging Network biorepository to MGH (n = 319) and Yale University (n = 344), processed, and sequenced on the NextSeq550. Reported fusions were manually reviewed, and novel fusions orthogonally verified via reverse-transcription PCR and Sanger sequencing., Results: AMP identified 148 fusions in 142/663 MOI-negative patients (21% [95% CI, 18 to 25]), of which 28 were covered by the Oncomine Comprehensive Assay (OCA) panel but missed, while 120 were not covered by OCA. Among AMP-identified positive patients, 32 had actionable fusions, 24 contained novel fusions, and six had two fusion events. We identified fusions in 12/34 (35% [95% CI, 20 to 54]) cholangiocarcinomas and 43/109 (39% [95% CI, 30 to 49]) sarcomas., Conclusion: Technology and awareness of actionable fusions have improved since the NCI-MATCH trial. With AMP-based technology, we identified 142 patients with fusions not detected during NCI-MATCH screening, many potentially actionable. These striking data underscore the need to optimize the fusion-detection capabilities of genotyping assays used in precision medicine.

Background: Cervical cancer is the most common genital cancer worldwide and is mainly caused by a persistent human papillomavirus infection. Well-known prognostic factors are age, histology, stage, stromal invasion, tumor size, and tumor grade. The relationship between the ABO and Rh system with cervical cancer has been studied since the 1950s, though without obtaining clear results. Here we investigated the association between the ABO blood group and Rh system and consecutively treated cervical cancer patients in our department. Methods: Clinical charts of cervical cancer patients treated and followed from 2010 to 2021 were checked for inclusion and exclusion criteria. Clinical and pathological data were recorded in a separate, anonymous, password-protected electronic database. All relevant data were extrapolated and used for final analysis. Results: A population of 143 cervical cancer patients was analyzed in this study. 47.6% (68/143) were blood group O, 36.4% (52/143) were blood group A, 8.4% (12/143) were blood group AB, and 7.7% (11/143) were blood group B. 14.9% (21/141) were RhD negative, while 85.1% (120/141) were RhD positive. No significant association was found between the ABO group and survival. However, patients with blood types B and AB had a higher BMI than the other blood types. RhD-negative patients exhibited a lower age at diagnosis (P=0.035) and had a higher overall survival compared to RhD-positive patients. Conclusions: The RhD factor appears to influence cervical cancer OS, but the data are too weakly significant to draw a definitive conclusion. Further studies with larger samples are needed to confirm this finding and to investigate the true impact of blood groups in female cancers., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Androgens are produced by adrenal and gonadal cells thanks to the action of specific enzymes. We investigated the role of protein kinase B (Akt) in the modulation of Δ4 steroidogenic enzymes' activity, in the mouse Leydig tumor cell line mLTC1. Cells were treated for 0-24 h with the 3 × 50% effective concentration of human luteinizing hormone (LH) and choriogonadotropin (hCG), in the presence and in the absence of the specific Akt inhibitor 3CAI. Cell signaling analysis was performed by bioluminescence resonance energy transfer (BRET) and Western blotting, while the expression of key target genes was investigated by real-time PCR. The synthesis of progesterone, 17α-hydroxy (OH)-progesterone and testosterone was measured by immunoassay. Control experiments for cell viability and caspase 3 activation were performed as well. We found that both hormones activated cAMP and downstream effectors, such as extracellularly-regulated kinase 1/2 (Erk1/2) and cAMP response element-binding protein (Creb), as well as Akt, and the transcription of Stard1, Hsd3b1, Cyp17a1 and Hsd17b3 genes, boosting the Δ4 steroidogenic pathway. Interestingly, Akt blockade decreased selectively Cyp17a1 expression levels, inhibiting its 17,20-lyase, but not the 17-hydroxylase activity. This effect is consistent with lower Cyp17a1 affinity to 17α-OH-progesterone than progesterone. As a result, cell treatment with 3CAI resulted in 17α-OH-progesterone accumulation at 16-24 h and decreased testosterone levels after 24 h. In conclusion, in the mouse Leydig cell line mLTC1, we found substantial Akt dependence of the 17,20-lyase activity and testosterone synthesis. Our results indicate that different intracellular pathways modulate selectively the dual activity of Cyp17a1., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: The advancement of Assisted Reproductive Technologies and the improvement in sperm freezing made male fertility preservation widely available. This study aims to evaluate the impact of cancer diseases on semen parameters before cryopreservation and the reproductive outcomes of patients who have thawed their semen samples., Methods: An observational, cohort study was conducted on cancer patients submitted to fertility preservation in AUSL-IRCCS of Reggio Emilia between 2007 and 2018. Semen samples were collected before cancer treatments, analyzed and frozen by rapid freezing. On request, these samples were thawed for Assisted Reproductive Technologies procedures. Semen parameters were compared between testicular versus other cancers., Results: We included 329 patients with a successful cryopreservation in 94.5% of cases. Testicular cancer was associated with lower sperm volumes (P=0.041) and lower total sperm concentration (P=0.009) compared to other cancers. No difference was observed about sperm motility and morphology, while oligozoospermia was significantly more frequent in men with testicular cancer (P<0.001). In our cohort, the 8.4% of patients thawed their samples; the usage rate and the embryo transfer rate were significantly higher (P<0.05) among those with a testicular cancer, while pregnancy and livebirth rates did not differ., Conclusions: Male fertility preservation is feasible, easy to be performed, non-invasive and does not delay cancer treatments. Men affected by testicular cancer had worse semen parameters at cryopreservation but pregnancy and livebirth rates were similar to those achieved by men with other cancers and similar to those achieved with fresh sperm.

Study Question: Does LH addition to FSH in vitro recover the human primary granulosa lutein cell (hGLC) sub/poor-response?, Summary Answer: A picomolar concentration of LH may recover the FSH-induced cAMP and progesterone production of hGLC from sub/poor-responder women., What Is Known Already: Clinical studies suggested that FSH and LH co-treatment may be beneficial for the ovarian response of sub/poor-responders undergoing ovarian stimulation during ART., Study Design, Size, Duration: hGLC samples from 286 anonymous women undergoing oocyte retrieval for ART were collected from October 2017 to February 2021., Participants/materials, Setting, Methods: hGLCs from women undergoing ovarian stimulation during ART were blindly purified, cultured, genotyped and treated in vitro by increasing concentrations of FSH (nM) ±0.5 nM LH. cAMP and progesterone levels produced after 3 and 24 h, respectively, were measured. In vitro data were stratified a posteriori, according to the donors' ovarian response, into normo-, sub- and poor-responder groups and statistically compared. The effects of LH addition to FSH were compared with those obtained by FSH alone in all the groups as well., Main Results and the Role of Chance: hGLCs from normo-responders were shown to have higher sensitivity to FSH treatment than sub-/poor-responders in vitro. Equimolar FSH concentrations induced higher cAMP (about 2.5- to 4.2-fold), and progesterone plateau levels (1.2- to 2.1-fold), in cells from normo-responder women than those from sub-/poor-responders (ANOVA; P < 0.05). The addition of LH to the cell treatment significantly increased overall FSH efficacy, indicated by cAMP and progesterone levels, within all groups (P > 0.05). Interestingly, these in vitro endpoints, collected from the normo-responder group treated with FSH alone, were similar to those obtained in the sub-/poor-responder group under FSH + LH treatment. No different allele frequencies and FSH receptor (FSHR) gene expression levels between groups were found, excluding genetics of gonadotropin and their receptors as a factor linked to the normo-, sub- and poor-response. In conclusion, FSH elicits phenotype-specific ovarian lutein cell response. Most importantly, LH addition may fill the gap between cAMP and steroid production patterns between normo- and sub/poor-responders., Limitations, Reasons for Caution: Although the number of experimental replicates is overall high for an in vitro study, clinical trials are required to demonstrate if the endpoints evaluated herein reflect parameters of successful ART. hGLC retrieved after ovarian stimulation may not fully reproduce the response to hormones of granulosa cells from the antral follicular stage., Wider Implications of the Findings: This in vitro assay may describe the individual response to personalize ART stimulation protocol, according to the normo-, sub- and poor-responder status. Moreover, this in vitro study supports the need to conduct optimally designed, randomized clinical trials exploring the personalized use of LH in assisted reproduction., Study Funding/competing Interest(s): This study was supported by Merck KGaA. M.L. and C.C. are employees of Merck KGaA or of the affiliate Merck Serono SpA. Other authors have no competing interests to declare., Trial Registration Number: N/A., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: The predictive role of sperm motility and morphology was recently detected in a large sample of more than 20000 assisted reproductive technology (ART) fresh cycles. However, the complete ART procedure consisted of both fresh and frozen-embryos transfers and only a comprehensive evaluation of the entire process could really confirm if these parameters really predict the ART success. The aim of the study was to evaluate which sperm parameter could predict the success of ART., Methods: A retrospective, real-world data analysis was performed, enrolling all couples attending ART from 2008 to 2021, including both fresh and frozen cycles and both in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI) procedures., Results: Fresh cycles success (considering live birth rate) was predicted by female age (1.04 [1.02-1.06]), injected oocytes (0.96 [0.93-0.99]), embryo number (0.79 [0.75-0.83]) and progressive sperm motility (0.98 [0.97-0.99]). On the contrary, frozen cycle outcomes were predicted only by sperm motility (0.97 [0.95-0.99]). This prediction was confirmed in IVF but not in ICSI cycles., Conclusion: Both female and male parameters predicted the ART success considering the entire path. However, frozen cycle success was predicted only by progressive sperm motility in IVF cycles, suggesting that the potential amelioration of this male parameter is relevant to improve ART success. Those couples expected to obtain the highest embryos after fertilization (low female age and better semen parameters) will have more attempts with frozen cycles and thus would benefit from a potential treatment focused to improve sperm parameters., (© 2022 American Society of Andrology and European Academy of Andrology.)

Uterine leiomyomas usually arise from the uterine body (95%), and rarely from the cervix (0.6%) or other urogenital sites. Lipoleiomyomas are benign, uncommon variants of leiomyomas (0.03-0.2%), histologically composed of smooth muscle cells and mature adipocytes; they usually occur in the uterine body and exceptionally in the cervix. We performed the first systematic literature review of cervical lipoleiomyomas (PRISMA guidelines), presenting five new cases. Including our series, thirty-one detailed cases were reported in the literature (mainly in Asia). The age range was 35-74 years, revealing a higher mean age than conventional cervical leiomyomas (46.5 vs. 39.4 years). Patients were usually multiparous (94%), typically complaining of vaginal bleeding (11/31, 36%), pelvic/abdominal pain (10/31, 32%), and/or urinary disturbances (6/31, 19%) 1 week to 10 months before presentation. Clinical examination revealed a pedunculated tumor (48%), or prolapse of ≥1 pelvic organs (16%). Twenty-four (77%) patients underwent total hysterectomy ± additional surgery; simple myomectomy/excision was performed in five (16%) cases. Only one (3%) of our cases recurred 2 years after partial excision; no evidence of disease was found 13 years after recurrence excision. Adipocytes occupied ≤50% of the tumor volume. Hyaline or myxoid changes and cartilaginous metaplasia were uncommon histological findings. Surgically challenging cases or pregnant patients may require expert gynecologists. Interventional radiology or conservative treatments were rarely proposed.

Primary vitreoretinal lymphoma (PVRL), a rare aggressive malignancy primarily involving the retina and/or the vitreous, is a major diagnostic challenge for clinicians (who commonly misdiagnose it as chronic uveitis) as well as for pathologists (for biological and technical reasons). Delays in diagnosis and treatment are responsible for visual impairments and life-threatening consequences, usually related to central nervous system involvement. The identification of lymphoma cells in vitreous fluid, obtained by vitrectomy, is required for diagnosis. Of note, the scarcity of neoplastic cells in small volumes of vitreous sample, and the fragility of lymphoma cells with degenerative changes caused by previous steroid use for presumed uveitis makes diagnosis based on cytology plus immunophenotyping difficult. Interleukin levels, immunoglobulin heavy chain or T-cell receptor gene rearrangements, and MYD88 mutation are applied in combination with cytology to support diagnosis. We aim to describe the current laboratory technologies for PVRL diagnosis, focusing on the main issues that these methods have. In addition, new emerging diagnostic strategies, such as next-generation sequencing analysis, are discussed. The genetic profile of PVRL remains largely unexplored. Better knowledge of genetic alterations is critical for precision medicine interventions with target-based treatments of this lymphoma for which no standardised treatment protocol currently exists.

Neoplasms with plasma cell differentiation may occasionally involve the skin. Cutaneous lesions may represent the first sign of an underlying systemic plasma cell malignancy, such as multiple myeloma, or the skin itself may be the primary site of occurrence of a hematological tumor with plasma cell differentiation. Starting from examples encountered in our daily practice, we discussed the diagnostic approach pathologists and clinicians should use when faced with cutaneous lesions with plasma cell differentiation. Cases of primary cutaneous marginal zone lymphoma, localized primary amyloidosis/amyloidoma, and cutaneous manifestations (secondary either to multiple myeloma or to plasmablastic lymphoma) are discussed, focusing on the importance of the adequate patient's work-up and precise clinicopathological correlation to get to the correct diagnosis and appropriate treatment. The pertinent literature has been reviewed, and the clinical presentation, pathological findings, main differential diagnoses, treatment, and outcome of neoplasms with plasma cell differentiation involving the skin are discussed.

Purpose: Several mathematical models have been developed to estimate individualized chances of assisted reproduction techniques (ART) success, although with limited clinical application. Our study aimed to develop a decisional algorithm able to predict pregnancy and live birth rates after controlled ovarian stimulation (COS) phase, helping the physician to decide whether to perform oocytes pick-up continuing the ongoing ART path., Methods: A single-center retrospective analysis of real-world data was carried out including all fresh ART cycles performed in 1998-2020. Baseline characteristics, ART parameters and biochemical/clinical pregnancies and live birth rates were collected. A seven-steps systematic approach for model development, combining linear regression analyses and decision trees (DT), was applied for biochemical, clinical pregnancy, and live birth rates., Results: Of fresh ART cycles, 12,275 were included. Linear regression analyses highlighted a relationship between number of ovarian follicles > 17 mm detected at ultrasound before pick-up (OF17), embryos number and fertilization rate, and biochemical and clinical pregnancy rates (p < 0.001), but not live birth rate. DT were created for biochemical pregnancy (statistical power-SP:80.8%), clinical pregnancy (SP:85.4%), and live birth (SP:87.2%). Thresholds for OF17 entered in all DT, while sperm motility entered the biochemical pregnancy's model, and female age entered the clinical pregnancy and live birth DT. In case of OF17 < 3, the chance of conceiving was < 6% for all DT., Conclusion: A systematic approach allows to identify OF17, female age, and sperm motility as pre-retrieval predictors of ART outcome, possibly reducing the socio-economic burden of ART failure, allowing the clinician to perform or not the oocytes pick-up., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: An explosive increase in couples attending assisted reproductive technology has been recently observed, despite an overall success rate of about 20%-30%. Considering the assisted reproductive technology-related economic and psycho-social costs, the improvement of these percentages is extremely relevant. However, in the identification of predictive markers of assisted reproductive technology success, male parameters are largely underestimated so far., Study Design: Retrospective, observational study., Objectives: To evaluate whether conventional semen parameters could predict assisted reproductive technology success., Materials and Methods: All couples attending a single third-level fertility center from 1992 to 2020 were retrospectively enrolled, collecting all semen and assisted reproductive technology parameters of fresh cycles. Fertilization rate was the primary end-point, representing a parameter immediately dependent on male contribution. Pregnancy and live birth rates were considered in relation to semen variables. Statistical analyses were performed using the parameters obtained according to the World Health Organization manual editions used for semen analysis., Results: Note that, 22,013 in vitro fertilization and intracytoplasmic sperm injection cycles were considered. Overall, fertilization rate was significantly lower in patients with abnormal semen parameters compared to normozoospermic men, irrespective of the World Health Organization manual edition. In the in vitro fertilization setting, both progressive motility (p = 0.012) and motility after capacitation (p = 0.002) significantly predicted the fertilization rate (statistical accuracy = 71.1%). Sperm motilities also predicted pregnancy (p < 0.001) and live birth (p = 0.001) rates. In intracytoplasmic sperm injection cycles, sperm morphology predicted fertilization rate (p = 0.001, statistical accuracy = 90.3%). Sperm morphology significantly predicted both pregnancy (p < 0.001) and live birth (p < 0.001) rates and a cut-off of 5.5% was identified as a threshold to predict clinical pregnancy (area under the curve = 0.811, p < 0.001)., Discussion: Interestingly, sperm motility plays a role in predicting in vitro fertilization success, while sperm morphology is the relevant parameter in intracytoplasmic sperm injection cycles. These parameters may be considered reliable tools to measure the male role on ART outcomes, potentially impacting the clinical management of infertile couples., (© 2021 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

Pembrolizumab (anti-PD-1) is allowed in selected metastatic castration-resistant prostate cancer (PC) patients showing microsatellite instability/mismatch repair system deficiency (MSI-H/dMMR). BRCA1/2 loss-of-function is linked to hereditary PCs and homologous recombination DNA-repair system deficiency: poly-ADP-ribose-polymerase inhibitors can be administered to BRCA -mutated PC patients. Recently, docetaxel-refractory metastatic castration-resistant PC patients with BRCA1/2 or ATM somatic mutations had higher response rates to pembrolizumab. PTEN regulates cell cycle/proliferation/apoptosis through pathways including the AKT/mTOR, which upregulates PD-L1 expression in PC. Our systematic literature review (PRISMA guidelines) investigated the potential correlations between PD-L1 and MMR/MSI/ BRCA/PTEN statuses in PC, discussing few other relevant genes. Excluding selection biases, 74/677 (11%) PCs showed dMMR/MSI; 8/67 (12%) of dMMR/MSI cases were PD-L1+. dMMR-PCs included ductal (3%) and acinar (14%) PCs (all cases tested for MSI were acinar-PCs). In total, 15/39 (39%) PCs harbored BRCA1/2 aberrations: limited data are available for PD-L1 expression in these patients. 13/137 (10%) PTEN- PCs were PD-L1+; 10/29 (35%) PD-L1+ PCs showed PTEN negativity. SPOP mutations may increase PD-L1 levels, while the potential correlation between PD-L1 and ERG expression in PC should be clarified. Further research should verify how the efficacy of PD-1 inhibitors in metastatic castration-resistant PCs is related to dMMR/MSI, DNA-damage repair genes defects, or PD-L1 expression.

Liquid biopsy is an accessible, non-invasive diagnostic tool for advanced prostate cancer (PC) patients, potentially representing a real-time monitoring test for tumor evolution and response to treatment through the analysis of circulating tumor cells (CTCs) and exosomes. We performed a systematic literature review (PRISMA guidelines) to describe the current knowledge about PD-L1 expression in liquid biopsies of PC patients: 101/159 (64%) cases revealed a variable number of PD-L1+ CTCs. Outcome correlations should be investigated in larger series. Nuclear PD-L1 expression by CTCs was occasionally associated with worse prognosis. Treatment (abiraterone, enzalutamide, radiotherapy, checkpoint-inhibitors) influenced PD-L1+ CTC levels. Discordance in PD-L1 status was detected between primary vs. metastatic PC tissue biopsies and CTCs vs. corresponding tumor tissues. PD-L1 is also released by PC cells through soluble exosomes, which could inhibit the T cell function, causing immune evasion. PD-L1+ PC-CTC monitoring and genomic profiling may better characterize the ongoing aggressive PC forms compared to PD-L1 evaluation on primary tumor biopsies/prostatectomy specimens (sometimes sampled a long time before recurrence/progression). Myeloid-derived suppressor cells and dendritic cells (DCs), which may have immune-suppressive effects in tumor microenvironment, have been found in PC patients circulation, sometimes expressing PD-L1. Occasionally, their levels correlated to clinical outcome. Enzalutamide-progressing castration-resistant PC patients revealed increased PD-1+ T cells and circulating PD-L1/2+ DCs.

Epigenetic alterations (including DNA methylation or miRNAs) influence oncogene/oncosuppressor gene expression without changing the DNA sequence. Prostate cancer (PC) displays a complex genetic and epigenetic regulation of cell-growth pathways and tumor progression. We performed a systematic literature review (following PRISMA guidelines) focused on the epigenetic regulation of PD-L1 expression in PC. In PC cell lines, CpG island methylation of the CD274 promoter negatively regulated PD-L1 expression. Histone modifiers also influence the PD-L1 transcription rate: the deletion or silencing of the histone modifiers MLL3/MML1 can positively regulate PD-L1 expression. Epigenetic drugs (EDs) may be promising in reprogramming tumor cells, reversing epigenetic modifications, and cancer immune evasion. EDs promoting a chromatin-inactive transcriptional state (such as bromodomain or p300/CBP inhibitors) downregulated PD-L1, while EDs favoring a chromatin-active state (i.e., histone deacetylase inhibitors) increased PD-L1 expression. miRNAs can regulate PD-L1 at a post-transcriptional level. miR-195/miR-16 were negatively associated with PD-L1 expression and positively correlated to longer biochemical recurrence-free survival; they also enhanced the radiotherapy efficacy in PC cell lines. miR-197 and miR-200a-c positively correlated to PD-L1 mRNA levels and inversely correlated to the methylation of PD-L1 promoter in a large series. miR-570, miR-34a and miR-513 may also be involved in epigenetic regulation.

The tumor microenvironment (TME) includes immune (T, B, NK, dendritic), stromal, mesenchymal, endothelial, adipocytic cells, extracellular matrix, and cytokines/chemokines/soluble factors regulating various intracellular signaling pathways (ISP) in tumor cells. TME influences the survival/progression of prostate cancer (PC), enabling tumor cell immune-evasion also through the activation of the PD-1/PD-L1 axis. We have performed a systematic literature review according to the PRISMA guidelines, to investigate how the PD-1/PD-L1 pathway is influenced by TME and ISPs. Tumor immune-escape mechanisms include suppression/exhaustion of tumor infiltrating cytotoxic T lymphocytes, inhibition of tumor suppressive NK cells, increase in immune-suppressive immune cells (regulatory T, M2 macrophagic, myeloid-derived suppressor, dendritic, stromal, and adipocytic cells). IFN-γ (the most investigated factor), TGF-β, TNF-α, IL-6, IL-17, IL-15, IL-27, complement factor C5a, and other soluble molecules secreted by TME components (and sometimes increased in patients' serum), as well as and hypoxia, influenced the regulation of PD-L1. Experimental studies using human and mouse PC cell lines (derived from either androgen-sensitive or androgen-resistant tumors) revealed that the intracellular ERK/MEK, Akt-mTOR, NF-kB, WNT and JAK/STAT pathways were involved in PD-L1 upregulation in PC. Blocking the PD-1/PD-L1 signaling by using immunotherapy drugs can prevent tumor immune-escape, increasing the anti-tumor activity of immune cells.

Many studies have investigated the potential prognostic and predictive role of PD-L1 in prostatic carcinoma (PC). We performed a systematic literature review (PRISMA guidelines) to critically evaluate human tissue-based studies (immunohistochemistry, molecular analysis, etc.), experimental research (cell lines, mouse models), and clinical trials. Despite some controversial results and study limitations, PD-L1 expression by tumor cells may be related to clinic-pathologic features of adverse outcome, including advanced tumor stage (high pT, presence of lymph node, and distant metastases), positivity of surgical margins, high Grade Group, and castration resistance. Different PD-L1 positivity rates may be observed in matched primary PCs and various metastatic sites of the same patients. Over-fixation, type/duration of decalcification, and PD-L1 antibody clone may influence the immunohistochemical analysis of PD-L1 on bone metastases. PD-L1 seemed expressed more frequently by castration-resistant PCs (49%) as compared to hormone-sensitive PCs (17%). Some series found that PD-L1 positivity was associated with decreased time to castration resistance. Treatment with ipilimumab, cyclophosphamide/GVAX/degarelix, or degarelix alone may increase PD-L1 expression. Correlation of PD-L1 positivity with overall survival and outcomes related to tumor recurrence were rarely investigated; the few analyzed series produced conflicting results and sometimes showed limitations. Further studies are required. The testing and scoring of PD-L1 should be standardized.

Immunotherapy targeting the PD-1-PD-L1 axis yielded good results in treating different immunologically ''hot'' tumors. A phase II study revealed good therapeutic activity of pembrolizumab in selected prostatic carcinoma (PC)-patients. We performed a systematic literature review (PRISMA guidelines), which analyzes the immunohistochemical expression of PD-L1 in human PC samples and highlights the pre-analytical and interpretation variables. Interestingly, 29% acinar PCs, 7% ductal PCs, and 46% neuroendocrine carcinomas/tumors were PD-L1+ on immunohistochemistry. Different scoring methods or cut-off criteria were applied on variable specimen-types, evaluating tumors showing different clinic-pathologic features. The positivity rate of different PD-L1 antibody clones in tumor cells ranged from 3% (SP142) to 50% (ABM4E54), excluding the single case tested for RM-320. The most tested clone was E1L3N, followed by 22C3 (most used for pembrolizumab eligibility), SP263, SP142, and 28-8, which gave the positivity rates of 35%, 11-41% (depending on different scoring systems), 6%, 3%, and 15%, respectively. Other clones were tested in <200 cases. The PD-L1 positivity rate was usually higher in tumors than benign tissues. It was higher in non-tissue microarray specimens (41-50% vs. 15%), as PC cells frequently showed heterogenous or focal PD-L1-staining. PD-L1 was expressed by immune or stromal cells in 12% and 69% cases, respectively. Tumor heterogeneity, inter-institutional preanalytics, and inter-observer interpretation variability may account for result biases.

In prostate cancer (PC), the PD-1/PD-L1 axis regulates various signaling pathways and it is influenced by extracellular factors. Pre-clinical experimental studies investigating the effects of various treatments (alone or combined) may discover how to overcome the immunotherapy-resistance in PC-patients. We performed a systematic literature review (PRISMA guidelines) to delineate the landscape of pre-clinical studies (including cell lines and mouse models) that tested treatments with effects on PD-L1 signaling in PC. NF-kB, MEK, JAK, or STAT inhibitors on human/mouse, primary/metastatic PC-cell lines variably down-modulated PD-L1-expression, reducing chemoresistance and tumor cell migration. If PC-cells were co-cultured with NK, CD8+ T-cells or CAR-T cells, the immune cell cytotoxicity increased when PD-L1 was downregulated (opposite effects for PD-L1 upregulation). In mouse models, radiotherapy, CDK4/6-inhibitors, and RB deletion induced PD-L1-upregulation, causing PC-immune-evasion. Epigenetic drugs may reduce PD-L1 expression. In some PC experimental models, blocking only the PD-1/PD-L1 pathway had limited efficacy in reducing the tumor growth. Anti-tumor effects could be increased by combining the PD-1/PD-L1 blockade with other approaches (inhibitors of tyrosine kinase, PI3K/mTOR or JAK/STAT3 pathways, p300/CBP; anti-RANKL and/or anti-CTLA-4 antibodies; cytokines; nitroxoline; DNA/cell vaccines; radiotherapy/Radium-223).

In IVF centers, risk assessment applies to complex processes potentially accounting for adverse events and reactions that undergo well-established legislative oversight, and nonconformances (NCs), that lack of established tracking systems. NCs account for an integral part of the quality management system, so that their documentation is important. The study evaluated the performance of a customized tool for incident reporting (IR) to track and characterize NCs in a public IVF center. IVF operators used the IVF-customized IR tool to record NCs at the moment of detection or subsequently, and in a time-saving manner during daily practice. From February 2015 to February 2020, 635 NCs were reported leading to the implementation of 10 operative instructions and 3 procedures with corrective strategies. NCs referred to the IVF laboratory were the most numerically meaningful (454/635, 71.5%). The majority (352/454, 77.5%) accounted for NCs related to procedures of sample management; considering the analytical phase as all the procedures involving sample treatment, the intra-analytical phase (176/352, 50%) has always been more subject to NCs compared to pre- (102/352, 29%) and postanalytical (74/352, 21%) phases. Our experience showed that the IVF-customized IR tool is suitable for application in IVF with regard to NC reports and documentation, as it identifies the most vulnerable steps of treatments. It manages NCs over the time, but it requires a contextual understanding of its application in order to avoid NC underestimates that could negatively influence the safety and quality aspects of IVF treatments., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Daria Morini et al.)

Background: A causative relationship between varicocele and impairment of semen quality has been largely investigated in the context of male infertility, although its clinical benefit remains controversial., Objective: To investigate the effect of varicocele correction on detailed morphologic microscopic semen parameters in a large homogeneous cohort of patients and to evaluate which factors could predict semen improvement after the surgical treatment., Materials and Methods: An observational, retrospective cohort study was carried out including all patients undergoing surgical treatment for varicocele from September 2011 to March 2020 in the same clinical centre. Enrolled males performed at least one semen analysis before and one after surgical varicocele correction. Primary outcome was the detailed morphologic microscopic sperm evaluation. Secondary outcomes were conventional semen analyses., Results: A total of 121 males (mean age 24.6 ± 6.1 years) were enrolled. Using detailed morphologic microscopic sperm evaluation, a significant morphological improvement was recorded, with a reduction in head and tail abnormalities. Moreover, a significant increase in sperm concentration (p = 0.015) and percentage of progressive and total motility (p = 0.022 and p = 0.039) were observed after surgery. The multivariate logistic analysis identified the ultrasonography varicocele degree before surgery as a main predictor of the sperm concentration improvement (p = 0.016), with the highest improvement for varicocele of I and II degree., Discussion: For the first time, the detailed morphologic microscopic sperm evaluation highlights a relevant reduction in sperm abnormalities after varicocele surgery, showing its potential application in clinical practice., (© 2021 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

Commercial hMG drugs are marketed for the treatment of infertility and consist of highly purified hormones acting on receptors expressed in target gonadal cells. Menopur® and Meriofert® are combined preparation of FSH and hCG and are compared in vitro herein. To this purpose, the molecular composition of the two drugs was analyzed by immunoassay. The formation of FSH receptor and LH/hCG receptor (FSHR; LHCGR) heteromer, intracellular Ca2+ and cAMP activation, β-arrestin 2 recruitment and the synthesis of progesterone and estradiol were evaluated in transfected HEK293 and human primary granulosa lutein cells treated by drugs administered within the pg-mg/ml concentration range. Molecular characterization revealed that Meriofert® has a higher FSH:hCG ratio than Menopur® which, in turn, displays the presence of LH molecules. While both drugs induced similar FSHR-LHCGR heteromeric formations and intracellular Ca2+ increase, Meriofert® had a higher potency than Menopur® in inducing a cAMP increase. Moreover, Meriofert® revealed a higher potency than Menopur® in recruiting β-arrestin 2, likely due to different FSH content modulating the tridimensional structure of FSHR-LHCGR-β-arrestin 2 complexes, as evidenced by a decrease in bioluminescence resonance energy transfer signal. This drug-specific activation of intracellular signaling pathways is consistent with the molecular composition of these preparations and impacts downstream progesterone and estradiol production, with Menopur® more potent than Meriofert® in inducing the synthesis of both the steroids. These findings are suggestive of distinct in-vivo activities of these preparations, but require cautious interpretation and further validation from clinical studies., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Classically, follicle-stimulating hormone receptor (FSHR)-driven cAMP-mediated signaling boosts human ovarian follicle growth and oocyte maturation. However, contradicting in vitro data suggest a different view on physiological significance of FSHR-mediated cAMP signaling. We found that the G-protein-coupled estrogen receptor (GPER) heteromerizes with FSHR, reprogramming cAMP/death signals into proliferative stimuli fundamental for sustaining oocyte survival. In human granulosa cells, survival signals are missing at high FSHR:GPER ratio, which negatively impacts follicle maturation and strongly correlates with preferential Gαs protein/cAMP-pathway coupling and FSH responsiveness of patients undergoing controlled ovarian stimulation. In contrast, FSHR/GPER heteromers triggered anti-apoptotic/proliferative FSH signaling delivered via the Gβγ dimer, whereas impairment of heteromer formation or GPER knockdown enhanced the FSH-dependent cell death and steroidogenesis. Therefore, our findings indicate how oocyte maturation depends on the capability of GPER to shape FSHR selective signals, indicating hormone receptor heteromers may be a marker of cell proliferation., Competing Interests: ML and GO are Merck Serono SpA employees without any conflict of interest., (© 2020 The Author(s).)

Establishing reliable prognostic factors as well as specific targets for new therapeutic approaches is an urgent requirement in advanced ovarian cancer. For several tumor entities, the ubiquitously spread scaffold protein β-arrestin 2, a multifunctional scaffold protein regulating signal transduction and internalization of activated G protein-coupled receptors (GPCRs), has been considered with rising interest for carcinogenesis. Therefore, we aimed to elucidate the prognostic impact of β-arrestin 2 and its functional role in ovarian cancer. β-arrestin 2 expression was analyzed in a subset of 156 samples of ovarian cancer patients by immunohistochemistry. Cytoplasmic expression levels were correlated with clinical as well as pathological characteristics and with prognosis. The biologic impact of β-arrestin 2 on cell proliferation and survival was evaluated, in vitro . Following transient transfection by increasing concentrations of plasmid encoding β-arrestin 2, different cell lines were evaluated in cell viability and death. β-arrestin 2 was detected in all histological ovarian cancer subtypes with highest intensity in clear cell histology. High β-arrestin 2 expression levels correlated with high-grade serous histology and the expression of the gonadotropin receptors FSHR and LHCGR, as well as the membrane estrogen receptor GPER and hCGβ. Higher cytoplasmic β-arrestin 2 expression was associated with a significantly impaired prognosis (median 29.88 vs. 50.64 months; P = 0.025). Clinical data were confirmed in transfected HEK293 cells, human immortalized granulosa cell line (hGL5) and the ovarian cancer cell line A2780 in vitro , where the induction of β-arrestin 2 cDNA expression enhanced cell viability, while the depletion of the molecule by siRNA resulted in cell death. Reflecting the role of β-arrestin 2 in modulating GPCR-induced proliferative and anti-apoptotic signals, we propose β-arrestin 2 as an important prognostic factor and also as a promising target for new therapeutic approaches in advanced ovarian cancer., (Copyright © 2020 Czogalla, Partenheimer, Jeschke, von Schönfeldt, Mayr, Mahner, Burges, Simoni, Melli, Benevelli, Bertini, Casarini and Trillsch.)