Analisi Genetiche e Molecolari di Tumori Eredo-Familiari

I tumori eredo-familiari insorgono a causa di una mutazione genetica trasmessa dai genitori e ciascun tumore ha il 5-10% di possibilità di avere un’origine ereditaria. Negli ultimi anni, l’applicazione della tecnologia Next Generation Sequencing (NGS) ha facilitato l’analisi attraverso l’utilizzo di pannelli multi genici. La diagnosi precoce del cancro e la consulenza genetica sono fondamentali affinché i pazienti o i parenti a rischio di sviluppare una malattia ereditaria possano conoscere la malattia e la sua trasmissione. Inoltre, la caratterizzazione genetica del paziente potrebbe guidare lo sviluppo di strategie terapeutiche mirate che agiscano selettivamente su percorsi molecolari alterati. In questa tesi sono analizzate mediante test molecolari varianti genetiche di patologie ereditarie tra cui: Melanoma Familiare (FM), Feocromocitoma e Paraganglioma (PPGL) e Sindrome di Lynch (LS). Il melanoma è un tumore maligno della pelle che deriva dalla trasformazione neoplastica e dalla proliferazione incontrollata dei melanociti. Il FM rappresenta quasi il 10% di questi tumori ed è spesso sotto diagnosticato. Per più di un decennio, solo le varianti nei geni CDKN2A e CDK4, coinvolti nel ciclo cellulare, sono state costantemente associate alla suscettibilità a FM. Negli ultimi anni sono stati identificati altri geni che predispongono al rischio di sviluppare melanoma come POT1, ACD, TERF2IP, TERT, BAP1, MITF, MC1R. Lo studio si propone di valutare l'utilizzo di un pannello NGS custom per la ricerca delle varianti nei pazienti che presentano un fattore genetico predisponente al melanoma e di analizzare la prevalenza delle varianti legate al territorio per avere una stima del rischio più accurata nell'area geografica. L'introduzione dell’analisi NGS per FM ha mostrato una Detection Rate (DR) del 15,07%. Considerando il confronto tra il test NGS e il test Sanger, quest’ultimo ha rivelato avere una sensibilità inferiore al 10% rispetto al pannello. I PPGL sono tumori neuroendocrini che originano dalla cresta neurale e che derivano dalle cellule cromaffini della midollare del surrene e dei paragangli. L'incidenza annuale è di circa due casi per milione, con la massima incidenza tra la terza e la quinta decade di età. I PPGL sono tumori ereditari e circa il 30% dei pazienti presenta una variante germinale in grado di predisporre alla malattia. Questo studio ha utilizzato il pannello NGS Clinical Exome Trusight One Combo (Illumina) per rilevare la presenza di varianti germinali nei geni correlati a PPGL. Lo studio ha incluso 23 pazienti con una DR del 21,7 % di varianti patogenetiche. La LS è la patologia tumorale ereditaria più comune; si tratta di una malattia genetica autosomica dominante associata ad un alto rischio di sviluppare tumori del colon-retto e dell'endometrio. La LS è il risultato di varianti patogenetiche della linea germinale nei geni del DNA Missmatch Repair (MMR). In questo percorso clinico/diagnostico è stata costruita una flow chart decisionale basata su test molecolari e consulenza genetica per rilevare nei pazienti la presenza di varianti ereditarie. L’obiettivo è individuare precocemente la presenza di varianti germinali per coinvolgere nello screening e nella consulenza genetica anche i familiari dei pazienti. I pazienti sospetti sono stati testati per l'instabilità dei microsatelliti (MSI), l'immunoistochimica per il MMR (IHC) e il test Multiple Ligation Probe Assay (MLPA). I risultati di questi primi screening hanno mostrato una corte di pazienti per il 23,6% Wild Type (WT) e indicati per il test NGS per ricercare alterazioni germinali mediante il pannello Hereditary Cancer (Illumina). Il test NGS effettuato sui pazienti WT selezionati ha mostrato il 20 % di varianti germinali patogenetiche nei geni del MMR.Hereditary cancer syndromes are a group of diseases that onset due to a genetic mutation transmitted by parents. Each cancer type has a 5-10% possibility of having a hereditary origin. In recent years, the application of Next Generation Sequencing (NGS) technology has facilitated multigene panel analysis. Early cancer detection and genetic counselling are essential for patients or relatives at risk of developing a disease with a potential hereditary component to know the nature of the genetic disorder and its transmission. Furthermore, genetic characterization of the patient at the germline level could guide future development of targeted therapeutic strategies that selectively act on altered molecular pathways. In this thesis are analysed genetic variants of hereditary pathologies of Familiar Melanoma (FM), Pheochromocytoma/Paraganglioma (PPGL) and Lynch Syndrome (LS) using molecular test and sequencing. Melanoma is a malignant skin tumour that results from the neoplastic transformation and uncontrolled proliferation of melanocytes. FM accounts for nearly 10% of these cancers and it is often underdiagnosed. For more than a decade, only variants in CDKN2A and CDK4 genes had been consistently associated with hereditary melanoma susceptibility. In recent years, other genes predisposing to the risk of developing melanoma have been identified, such as POT1, ACD, TERF2IP, TERT, BAP1, MITF, MC1R. The study aims to evaluate the impact of NGS custom panel in patients that have a genetic factor predisposing to melanoma and to analyse the prevalence of territory-related variants to have a more accurate risk estimation in the geographical area. The introduction of a NGS gene panel for FM as a germline testing strategy for melanoma patients showed a Detection Rate (DR) of 15.07%. If we consider the comparison between the NGS panel test and the Sanger standard test, the latter had less than 10% sensitivity compared to melanoma’s gene panel. PPGLs are neuroendocrine tumours arising from the neural crest and derive from the chromaffin cells of the adrenal medullary and paraganglia.The annual incidence of these rare tumours is approximately two cases per million, with the highest incidence between the third and fifth decades of age. PPGLs are hereditary tumours with around 30 % of patients that have a germline variant able to predispose to the tumour. It is a rare disease but has a high variability, with different symptoms that are frequently hidden in other clinical conditions. This study used the NGS Clinical Exome Trusight One Combo panel (Illumina) to detect the presence of germline variants in genes correlated to PPGL. The study included 23 patients and analysis showed a DR of 21.7 % pathogenic variants. LS is the most common inherited cancer syndrome; it is an autosomal dominant genetic disorder associated with a greatly increased risk of developing Colon Rectal cancers and Endometrial cancers. LS is the result of germline pathogenetic variants in DNA mismatch repair (MMR) genes. In this clinical and diagnostic pathway, a decisional flow chart was built based on molecular tests and genetic counselling to detect the presence of hereditary mutation in patients. This project aims to detect the presence of hereditary variants early to involve, eventually, patients’ family members in screening and genetic counselling. Patients were tested for microsatellite instability (MSI), MMR deficiency immunohistochemistry (IHC) and Multiple Ligation Probe Assay (MLPA). The results of these preliminary tests showed that 23.6% of patients was Wild Type (WT) and indicated for the NGS test to search for germinal variants by Hereditary Cancer panel (Illumina). The NGS test performed on the selected WT patients showed 20% pathogenic germline variants in MMR genes

Which sperm parameter limits could really guide the clinical decision in assisted reproduction?

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

Protein kinase B (Akt) blockade inhibits LH/hCG-mediated 17,20-lyase, but not 17α-hydroxylase activity of Cyp17a1 in mouse Leydig cell steroidogenesis

: 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

Semen cryopreservation in men undergoing cancer treatment: a ten-year study

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, analysed 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

β-arrestin 2 Is a Prognostic Factor for Survival of Ovarian Cancer Patients Upregulating Cell Proliferation

International audienceEstablishing 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 beta-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 beta-arrestin 2 and its functional role in ovarian cancer. beta-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 beta-arrestin 2 on cell proliferation and survival was evaluated,in vitro. Following transient transfection by increasing concentrations of plasmid encoding beta-arrestin 2, different cell lines were evaluated in cell viability and death. beta-arrestin 2 was detected in all histological ovarian cancer subtypes with highest intensity in clear cell histology. High beta-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 hCG beta. Higher cytoplasmic beta-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 A2780in vitro, where the induction of beta-arrestin 2 cDNA expression enhanced cell viability, while the depletion of the molecule by siRNA resulted in cell death. Reflecting the role of beta-arrestin 2 in modulating GPCR-induced proliferative and anti-apoptotic signals, we propose beta-arrestin 2 as an important prognostic factor and also as a promising target for new therapeutic approaches in advanced ovarian cancer

Lipoleiomyomas of the Uterine Cervix: A New Series including the First Recurrent Case and the First Systematic Literature Review

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

Two human menopausal gonadotrophin (hMG) preparations display different early signaling in vitro

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\uae and Meriofert\uae 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, \u3b2-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\uae has a higher FSH:hCG ratio than Menopur\uae which, in turn, displays the presence of LH molecules. While both drugs induced similar FSHR-LHCGR heteromeric formations and intracellular Ca2+ increase, Meriofert\uae had a higher potency than Menopur\uae in inducing a cAMP increase. Moreover, Meriofert\uae revealed a higher potency than Menopur\uae in recruiting \u3b2-arrestin 2, likely due to different FSH content modulating the tridimensional structure of FSHR-LHCGR-\u3b2-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\uae more potent than Meriofert\uae 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

Cutaneous Involvement in Diseases with Plasma Cell Differentiation: Diagnostic Approach

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

What Do We Have to Know about PD-L1 Expression in Prostate Cancer? A Systematic Literature Review. Part 7: PD-L1 Expression in Liquid Biopsy

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