Embryonal Carcinoma Research Articles

P-381 Fertility preservation in patients with testicular cancer

Abstract Study question Do the clinical stage and histological characteristics of testicular cancer affect sperm quality? Summary answer Sperm concentration, motility and normal morphology significantly decreased in patients with testicular cancer. However, semen parameters were not significantly associated with tumor histology. What is known already Testicular cancer is rare in most countries, with an incidence ranging from ∼1/100,000 to 10/100,000, and represents about 1% of all cancers in men, but about 60% of all cancers in young males aged between 15 and 35 years. Additionally, the incidence of testicular cancer has doubled in the last 20-40 years. While it is clear that chemotherapy treatment can negatively impact infertility, it is increasingly recognized that male-factor infertility may play a significant role in the overall health status of men, and men with infertility may be at higher risk of developing testicular cancer. Study design, size, duration Retrospective data from 284 males, diagnosed with testicular cancer, undergoing to semen cryopreservation before surgery or chemo- or radiotherapy treatments, from January 2016 to June 2022 at Maternal and Child Department, Gynecology and Obstetrics Unit, Couple Sterility Center at Federico II, University of Naples, were included in the study. Participants/materials, setting, methods All patients signed their informed consent to cryopreservation. In order to have an internal control group coming from the same region, results of semen analysis from male partners of couples with certain female factors (bilateral tubal imperviousness or in case of egg donation) were included in the study. After semen collection and liquefaction, the semen specimens were cryopreserved following manufacturer’s protocol with media conteining TEST-yolk buffer. Main results and the role of chance Overall, taking in to consideration the semen cryopreservation, per WHO 2021 criteria, we observed that the majority of semen parameters of patients with testicular cancer resulted between 5°percentile and 25° percentile. The unique parameter over the 50°percentile was the semen volume. Comparing results from male partners of couples with certain female factors and testicular cancer patients several features were significant different. Especially, semen volume resulted significantly increased in cases respect to the control (p = 0,011). Instead, from microscopic evaluation semen concentration, percentage of total sperm motility and % of spermatozoa with normal morphology appeared significantly decreased in patients with testicular cancer (p = 0,000; p = 0,000; p = 0,002). Based on Histopathological Classification of Testicular Tumors the 59% (168/284) were classified as classic seminoma, 14% (41/284) mixed germ cell tumors including seminomatous and nonseminomatous cell tumors, 13% (38/284) nonseminomatous mixed germ cell tumors, 12% (35/284) nonseminomatous germ cell tumors including 4 cases of teratoma and 30 cases of embryonal carcinoma and 2 cases of cariocarcinoma. However, abnormal semen parameters were not significantly associated with tumor histology. Limitations, reasons for caution No information about semen quality after thawing and ICSI outcome could represent the main limitation for a complete information of the preservation program in these patients. Wider implications of the findings We presented our monocentric experience of oncofertility preservation program with especially attention to testicular cancer patients. Presented data emphasizes the importance of semen monitoring in young men and the fertility preservation in management of the patient. Trial registration number not applicable

Usp7 Regulates Glial Lineage Cell-Specific Transcription Factors by Modulating Histone H2B Monoubiquitination.

Histone H2B monoubiquitination (H2Bub1) is a dynamic posttranslational modification which are linked to DNA damage and plays a key role in a wide variety of regulatory transcriptional programs. Cancer cells exhibit a variety of epigenetic changes, particularly any aberrant H2Bub1 has frequently been associated with the development of tumors. Nevertheless, our understanding of the mechanisms governing the histone H2B deubiquitination and their associated functions during stem cell differentiation remain only partially understood. In this study, we wished to investigate the role of deubiquitinating enzymes (DUBs) on H2Bub1 regulation during stem cell differentiation. In a search for potential DUBs for H2B monoubiquitination, we identified Usp7, a ubiquitin-specific protease that acts as a negative regulator of H2B ubiquitination during the neuronal differentiation of mouse embryonic carcinoma cells. Loss of function of the Usp7 gene by a CRISPR/Cas9 system during retinoic acid-mediated cell differentiation contributes to the increase in H2Bub1. Furthermore, knockout of the Usp7 gene particularly elevated the expression of neuronal differentiation related genes including astryocyte-specific markers and oligodendrocyte-specific markers. In particular, glial lineage cell-specific transcription factors including oligodendrocyte transcription factor 2, glial fibrillary acidic protein, and SRY-box transcription factor 10 was significantly upregulated during neuronal differentiation. Thus, our findings suggest a novel role of Usp7 in gliogenesis in mouse embryonic carcinoma cells.

Expression of Podocalyxin Potentially Decorated With Low-sulfated Keratan Sulfate in Human Testicular Embryonal Carcinoma.

SummaryWe previously demonstrated that among various histological types of human testicular germinal cell tumors (GCTs), embryonal carcinoma (EC) preferentially expresses low-sulfated keratan sulfate (KS) consisting of repeating N-acetyllactosamine (LacNAc) disaccharide units composed of galactose and 6-O-sulfated N-acetylglucosamine (GlcNAc), which is recognized by the R-10G antibody. Recently, we generated another anti-low-sulfated KS monoclonal antibody, 294-1B1. Immunohistochemical analysis of testicular GCTs (n=83) revealed that the low-sulfated KS recognized by 294-1B1 is also preferentially expressed in EC but minimally in other GCT histological types. Moreover, immunolabeling with R-10G and 294-1B1 antibodies was resistant to peptide-N-glycosidase F digestion, and EC was not stained with the MECA-79 antibody, indicating that low-sulfated KS expressed in EC contains mucin-type core 2 O-glycans carrying GlcNAc-6-O-sulfated oligo-LacNAc. Double immunofluorescence staining showed that R-10G and 294-1B1 antibody signals colocalized with those for podocalyxin (PODXL). Furthermore, western blot analysis of recombinant human PODXL•IgG fusion proteins secreted from low-sulfated KS-expressing human embryonic kidney 293T cells revealed that PODXL functions as a core protein for low-sulfated KS. Taken together, these findings strongly suggest that the PODXL glycoform decorated with low-sulfated KS is preferentially expressed in human testicular EC and may therefore serve as a diagnostic marker for this malignancy.

Pathogenesis and pathobiology of testicular germ cell tumours: a view from a developmental biological perspective with guidelines for pathological diagnostics.

Testicular germ cell tumours (GCT) are divided into three different subtypes (types I-III) regarding to their developmental origin, histological differences and molecular features. Type I GCT develop from disturbed primordial germ cells and most commonly occur in children and young adolescents, which is why they are referred to as prepubertal GCT. Type II GCT develop from a non-invasive germ cell neoplasia in situ (GCNIS) and show an isochromosome 12p (i12p) or gain of 12p material as a common and characteristic molecular alteration. Type III GCT originate from distorted postpubertal germ cells (e.g. spermatogonia) in adult patients and have changes on chromosome 9 with amplification of the DMRT1 gene. Type I GCT encompass prepubertal-type teratomas and yolk-sac tumours (YST). Type II GCT include seminoma, embryonal carcinoma, choriocarcinoma, postpubertal-type teratoma and postpubertal-type YST. Types I and II GCT both show similar morphology, but are separated from each other by the detection of a GCNIS and an i12p in type II GCT. For type II GCT it is especially important to detect non-seminomatous elements, as these tumours have a worse biological behaviour and need a different treatment to seminomas. In contrast to types I and II GCT, type III tumours are equivalent to spermatocytic tumours and usually occur in elderly men, with few exceptions in young adults. The development of types I and II GCT seems to depend not upon driver mutations, but rather on changes in the epigenetic landscape. Furthermore, different pluripotency associated factors (e.g. OCT3/4, SOX2, SOX17) play a crucial role in GCT development and can be used as immunohistochemical markers allowing to distinguish the different subtypes from each other in morphologically challenging tissue specimens. Especially in metastatic sites, a morphological and immunohistochemical diagnostic algorithm is important to detect small subpopulations of each non-seminomatous GCT subtype, which are associated with a poorer prognosis and need a different treatment. Furthermore, primary extragonadal GCT of the retroperitoneum or mediastinum develop from misguided germ cells during embryonic development, and might be challenging to detect in small tissue biopsies due to their rarity at corresponding sites. This review article summarises the pathobiological and developmental aspects of the three different types of testicular GCT that can be helpful in the histopathological examination of tumour specimens by pathologists.

Expression Proteomics and Histone Analysis Reveal Extensive Chromatin Network Changes and a Role for Histone Tail Trimming during Cellular Differentiation.

In order to understand the coordinated proteome changes associated with differentiation of a cultured cell pluripotency model, protein expression changes induced by treatment of NT2 embryonal carcinoma cells with retinoic acid were monitored by mass spectrometry. The relative levels of over 5000 proteins were mapped across distinct cell fractions. Analysis of the chromatin fraction revealed major abundance changes among chromatin proteins and epigenetic pathways between the pluripotent and differentiated states. Protein complexes associated with epigenetic regulation of gene expression, chromatin remodelling (e.g., SWI/SNF, NuRD) and histone-modifying enzymes (e.g., Polycomb, MLL) were found to be extensively regulated. We therefore investigated histone modifications before and after differentiation, observing changes in the global levels of lysine acetylation and methylation across the four canonical histone protein families, as well as among variant histones. We identified the set of proteins with affinity to peptides housing the histone marks H3K4me3 and H3K27me3, and found increased levels of chromatin-associated histone H3 tail trimming following differentiation that correlated with increased expression levels of cathepsin proteases. We further found that inhibition of cathepsins B and D reduces histone H3 clipping. Overall, the work reveals a global reorganization of the cell proteome congruent with differentiation, highlighting the key role of multiple epigenetic pathways, and demonstrating a direct link between cathepsin B and D activity and histone modification.

GCT-27. Β-HCG STIMULATION WITH RAPIDLY PROGRESSIVE PUBERTY (PP) IN INTRACRANIAL NON-GERMINOMATOUS GCT (NGGCT): CASE REPORT

Abstract BACKGROUND Intracranial non-germinomatous are rare heterogenous group of Central Nervous System (CNS) tumors that consist of embryonal carcinomas, yolk sac tumors, choriocarcinomas, and teratomas (mature, immature), with or without malignant transformation, as well as mixed types. Presentation frequently includes endocrinopathies, visual changes and signs of raised intracranial pressure. CASE We present a 9-year-old boy with history of headaches, voice changes, increased height and muscle mass, and new onset pubic hair for one month. Examination showed mild papilledema with no cranial nerve palsies, advanced linear growth with height velocity of 15.8 cm/yr with height and weight at 94th and 85th percentile respectively for age. Tanner 2 pubic hair, muscular body habitus and testicles volume of 10-12mLs bilaterally. MRI brain showed a localized pineal region mass with obstructive hydrocephalus. Tumor markers were elevated in serum (β-hCG;69 IU/l and AFP;36.8 ng/ml) with high CSF β-hCG of 41 IU/l with AFP <10ng/ml. Work up for advanced puberty showed bone age of 11.3yrs. Markedly elevated testosterone levels at 929 ng/dl with free testosterone of 135 pg/m. Both LH and FSH were undetectable supporting the diagnosis of peripheral non-gonadotropic puberty (PP). Diagnosis of NGGCT was established on biopsy. He received therapy with chemotherapy, second look surgery and radiation. He remains in remission (negative tumors markers and MRI). Interestingly initiation of therapy resulted in age-appropriate testosterone level and slowing down of rapidly progressive puberty clinically. CONCLUSION We herein present a rare association of β-hCG secreted by NGGCT that cross-reacts with testicular and adrenal LH receptors resulting in increased testosterone levels and rapidly progressive or precocious puberty in males. The incidence of PP in pineal NGGCT remains unknown with very few cases reported in the literature however these are an important consideration in children with advanced puberty in the absence of exogenous hormone exposure and or non-CNS etiologies.

Mouse embryos, chimeras, and embryonal carcinoma stem cells-Reflections on the winding road to gene manipulation.

The relationship of embryonal carcinoma (EC) cells, the stem cells of germ cell- or embryo-derived teratocarcinoma tumors, to early embryonic cells came under intense scrutiny in the early 1970s when mouse chimeras were produced between EC cells and embryos. These chimeras raised tantalizing possibilities and high hopes for different areas of research. The normalization of EC cells by the embryo lent validity to their use as in vitro models for embryogenesis and indicated that they might reveal information about the relationship between malignancy and differentiation. Chimeras also showed the way for the potential introduction of genes, selected in EC cells in vitro, into the germ line of mice. Although EC cells provided material for the elucidation of early embryonic events and stimulated many studies of early molecular differentiation, after years of intense scrutiny,they fell short as the means of genetic manipulation of the germ line, although arguably they pointed the way to the development of embryonic stem (ES) cells that eventually fulfilled this goal.

Molecular and epigenetic ex vivo profiling of testis cancer-associated fibroblasts and their interaction with germ cell tumor cells and macrophages

Germ cell tumors (GCT) are the most common solid tumors in young men of age 15 - 40. In previous studies, we profiled the interaction of GCT cells with cells of the tumor microenvironment (TM), which showed that especially the 3D interaction of fibroblasts (FB) or macrophages with GCT cells influenced the growth behavior and cisplatin response as well as the transcriptome and secretome of the tumor cells, suggesting that the crosstalk of these cells with GCT cells is crucial for tumor progression and therapy outcome.In this study, we shed light on the mechanisms of activation of cancer-associated fibroblasts (CAF) in the GCT setting and their effects on GCT cells lines and the monocyte cell line THP-1. Ex vivo cultures of GCT-derived CAF were established and characterized molecularly and epigenetically by performing DNA methylation arrays, RNA sequencing, and mass spectrometry-based secretome analysis.We demonstrated that the activation state of CAF is influenced by their former prevailing tumor environment in which they have resided. Hereby, we postulate that seminoma (SE) and embryonal carcinoma (EC) activate CAF, while teratoma (TER) play only a minor role in CAF formation. In turn, CAF influence proliferation and the expression of cisplatin sensitivity-related factors in GCT cells lines as well as polarization of in vitro-induced macrophages by the identified effector molecules IGFBP1, LGALS3BP, LYVE1, and PTX3.Our data suggests that the vital interaction of CAF with GCT cells and with macrophages has a huge influence on shaping the extracellular matrix as well as on recruitment of immune cells to the TM. In conclusion, therapeutically interfering with CAF and / or macrophages in addition to the standard therapy might slow-down progression of GCT and re-shaping of the TM to a tumor-promoting environment.Significance: The interaction of CAF with GCT and macrophages considerably influences the microenvironment. Thus, therapeutically interfering with CAF might slow-down progression of GCT and re-shaping of the microenvironment to a tumor-promoting environment.

Higher expression of SALL4-A isoform is correlated with worse outcomes and progression of the disease in subtype of testicular germ cell tumours

Background The transcription factor SALL4 is associated with embryonic pluripotency and has proposed as a novel immunohistochemistry (IHC) marker for diagnosing germ cell tumours. SALL4 comprises three isoforms, and SALL4-A being the full-length isoform. Studying its isoforms could revolutionize testicular cancer prognosis and subtype differentiation. Methods The expression and clinical significance of isoform ‘A’ of SALL4 was evaluated in 124 testicular germ cell tumours (TGCTs) subtypes, adjacent 67 normal tissues and 22 benign tumours, using immunohistochemistry on tissue microarrays (TMA). Results A statistically significant higher expression of nuclear and cytoplasmic SALL4-A was detected in TGCTs histological subtypes and benign tumours compared to the normal tissues. Seminoma and yolk sac tumours had the highest nuclear and cytoplasmic expression of SALL4-A. A significant correlation was detected between the higher nuclear expression of SALL4-A and increased pT stages (P = 0.026) in seminomas. Whereas in embryonal carcinomas, cytoplasmic expression of SALL4-A was associated with the tumour recurrence (P = 0.04) and invasion of the epididymis (P = 0.011). Conclusions SALL4-A isoform expression in the cytoplasm and nucleus of TGCTs may be associated with histological differentiation. In the seminoma subtype of TGCTs, higher expression of SALL4-A may be used as a predictive indicator of poorer outcomes and prognosis.

Propafenone-induced connexin-43 protein accumulation and intracellular dynamics

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): China Scholarship Council Background Connexin-43 (Cx43) plays a pivotal role in intercellular communication through gap junctions. It is instrumental in propagating electrical signals and allowing ions to flow between neighboring cardiomyocytes. This synchronized electrical activity allows the heart to work as a functional syncytium. Any disruption or malfunction of Cx43 can lead to arrhythmias and other cardiac issues. Understanding the functions and regulation of Cx43 is vital in both basic research and clinical contexts. Propafenone, a class Ic antiarrhythmic drug, has shown promise in rhythm control; however, its precise impact on cardiac cellular physiology, particularly regarding Cx43, remains incompletely understood. Purpose To investigate propafenone’s effect on Cx43 expression, physiology, and underlying mechanisms in cell systems. Methods Used cell lines include human embryonic kidney HEK293 cells transfected with Cx43; differentiated murine embryonic carcinoma EPI7 cells with an epithelioid morphology and visceral endoderm-like END2 cells, endogenously expressing high amounts of functional Cx43. Cx43 protein expression levels were determined by Western blot analysis, whereas immunofluorescence (IF) imaged by a Total Internal Reflection Fluorescence (TIRF) microscope was used to assess the subcellular localization of Cx43 proteins. Dye injections were used to gain insight into the effects of propafenone on Cx43 metabolic coupling. Results and Conclusion Full length Cx43 protein levels were increased after treatment with propafenone and IF microscopy showed an intracellular accumulation of Cx43 protein, both on ectopically and endogenously expressed Cx43. Propafenone did not alter Cx43 half-life, in contrast to the lysosomal inhibitor chloroquine. Finally, gap-junctional coupling was decreased by chronic propafenone treatment. We conclude that the class 1c antiarrhythmic drug propanone increases Cx43 protein expression, resulting in its intracellular accumulation, as a side effect.

Laccase-Treated Polystyrene Surfaces with Caffeic Acid, Dopamine, and L-3,4-Dihydroxyphenylalanine Substrates Facilitate the Proliferation of Melanocytes and Embryonal Carcinoma Cells NTERA-2.

This study presents the effects of treating polystyrene (PS) cell culture plastic with oxidoreductase enzyme laccase and the catechol substrates caffeic acid (CA), L-DOPA, and dopamine on the culturing of normal human epidermal melanocytes (NHEMs) and human embryonal carcinoma cells (NTERA-2). The laccase-substrate treatment improved PS hydrophilicity and roughness, increasing NHEM and NTERA-2 adherence, proliferation, and NHEM melanogenesis to a level comparable with conventional plasma treatment. Cell adherence dynamics and proliferation were evaluated. The NHEM endpoint function was quantified by measuring melanin content. PS surfaces treated with laccase and its substrates demonstrated the forming of polymer-like structures. The surface texture roughness gradient and the peak curvature were higher on PS treated with a combination of laccase and substrates than laccase alone. The number of adherent NHEM and NTERA-2 was significantly higher than on the untreated surface. The proliferation of NHEM and NTERA-2 correspondingly increased on treated surfaces. NHEM melanin content was enhanced 6-10-fold on treated surfaces. In summary, laccase- and laccase-substrate-modified PS possess improved PS surface chemistry/hydrophilicity and altered roughness compared to untreated and plasma-treated surfaces, facilitating cellular adherence, subsequent proliferation, and exertion of the melanotic phenotype. The presented technology is easy to apply and creates a promising custom-made, substrate-based, cell-type-specific platform for both 2D and 3D cell culture.

Prognostıc Factors In Testıcular Cancer

Aim: Testicular cancer is the most common cancer in men aged 15-35 years and accounts for 1% of all lifetime male cancers. There are two histologic subtypes: seminoma and nonseminoma. In our study, we aimed to investigate the factors predicting recurrence in early-stage testicular cancer. Materials and Methods: Our study is a retrospective study of early stage testicular cancer admitted to the medical oncololi clinic of our hospital between 2006-2018. During the study, 344 patient files were reviewed and 130 patients who met the study criteria were included in the study. Our primary aim in this study was to investigate the factors predictive of recurrence in early stage testicular cancer. Results: When evaluating PFS in patients with nonseminoma with and without lymphovascular invasion, no median PFS value was reached in either group. However, PFS was worse in patients with LVI (p=0.037). When comparing stage 1 with stage 2 seminoma patients, no median PFS values could be reached, but there was a statistical difference between the two groups in terms of recurrence (p=0.019). Conclusions: In our study, we found no association between tumor size, embryonal carcinoma predominance, tunica albuginea invasion, spermatic cord involvement and tumor marker values and recurrence in nonseminoma germ cell testicular tumors. PFS was shorter in patients with LVI compared to those without LVI. Lymphovascular invasion, spermatic cord involvement, tunica albuginea involvement, and rete testis involvement were not associated with disease recurrence in seminoma patients, whereas higher disease stage predicted the risk of recurrence.

Comprehensive microRNA expression analysis of pediatric gonadal germ cell tumors: unveiling novel biomarkers and signatures.

microRNAs (miRNAs) are small endogenous noncoding RNAs, and alterations in their expression may contribute to oncogenesis. Discovering a unique miRNA pattern holds the potential for early detection and novel treatment possibilities in cancer. This study aimed to evaluate miRNA expression in pediatric patients with gonadal germ cell tumors (GCTs), focusing on characterizing the miRNA profiles of each histological subtype and identifying a distinct histological miRNA signature for a total of 42 samples of pediatric gonadal GCTs. The analysis revealed distinct miRNA expression profiles for all histological types, regardless of the primary site. We identified specific miRNA expression signatures for each histological type, including 34 miRNAs for dysgerminomas, 13 for embryonal carcinomas, 25 for yolk sac tumors, and one for immature teratoma, compared to healthy controls. Furthermore, we identified 26 miRNAs that were commonly expressed in malignant tumors, with six miRNAs (miR-302a-3p, miR-302b-3p, miR-371a-5p, miR-372-3p, miR-373-3p, and miR-367-3p) showing significant overexpression. Notably, miR-302b-3p exhibited a significant association with all the evaluated clinical features. Our findings suggest that miRNAs have the potential to aid in the diagnosis, prognosis, and management of patients with malignant GCTs.

Metastatic germ cell tumors in serous cavities and cerebrospinal fluid: A single-center experience.

Metastatic germ cell tumors (GCTs) involving body cavity effusions and cerebrospinal fluid (CSF) are rare. Diagnosis is challenging because of limited morphological and clinicopathological information in the literature. A database search of our institution from 1990 to 2024 identified 27 cases of metastatic GCTs, comprising five pediatric and 22 adolescent and adult patients, in serous cavities or the CSF, including peritoneal (15), pleural (nine), CSF (two), and pericardial (one) fluid. The most common primary site was the testis (n=10), followed by the ovaries (n=7), mediastinum (n=4), retroperitoneum (n=3), pineal gland (n=2), and sacrum/coccyx (n=1). The primary tumors in 14 patients were mixed GCTs (six with a seminoma component), followed by immature teratomas (six), yolk sac tumors (three), embryonal carcinomas (two), pure seminomas (one), and postpubertal teratomas (one). The median interval between primary tumor diagnosis and diagnosis of fluid positivity was 7 months (range: 0-134 months). In nine cases, the malignant fluid was diagnosed simultaneously with or within 1 month of the primary tumor. GCT subtyping was performed on 23 of the 27 cytological specimens. Twenty-four patients (89%) also had metastases to other sites. Thirteen patients died of the disease (48%), with a median survival time of 4 months. Metastatic GCTs in serous effusions and CSF are often associated with disseminated disease and poor prognosis. Subtyping can be performed by cytomorphology combined with immunohistochemistry.

Pathological and Biological Significance of the Specific Glycan, TRA-1-60, on Aggressive Gastric Adenocarcinoma

The glycans form a unique complex on the surface of cancer cells and play a pivotal role in tumor progression, impacting proliferation, invasion, and metastasis. TRA-1-60 is a glycan that was identified as a critical marker for the establishment of fully reprogrammed inducible pluripotent stem cells. Its expression has been detected in multiple cancer tissues, including embryonal carcinoma, prostate cancer, and pancreatic cancer, but the biological and pathological characterization of TRA-1-60-expressing tumor cells remains unclear within various types of malignancies. Here, we report the biological characteristics of TRA-1-60-expressing gastric cancer cells, especially those with its cell surface expression, and the therapeutic significance of targeting TRA-1-60. The cells with cell membrane expression of TRA-1-60 were mainly observed in the invasive area of patient gastric cancer tissues and correlated with advanced stages of the disease based on histopathological and clinicopathological analyses. In vitro analysis using a scirrhous gastric adenocarcinoma line, HSC-58, which highly expresses TRA-1-60 on its plasma membrane, revealed increased stress-resistant mechanisms, supported by the upregulation of glutathione synthetase and NCF-1 (p47phox) via lipid–ROS regulatory pathways, as detected by RNA-seq analysis followed by oxidative stress gene profiling. Our in vivo therapeutic study using the TRA-1-60-targeting antibody–drug conjugate, namely, Bstrongomab-conjugated monomethyl auristatin E, showed robust efficacy in a mouse model of peritoneal carcinomatosis induced by intraperitoneal xenograft of HSC-58, by markedly reducing massive tumor ascites. Thus, targeting the specific cell surface glycan, TRA-1-60, shows a significant therapeutic impact in advanced-stage gastric cancers.

NANOG controls testicular germ cell tumour stemness through regulation of MIR9-2.

Testicular germ cell tumours (TGCTs) represent a clinical challenge; they are most prevalent in young individuals and are triggered by molecular mechanisms that are not fully understood. The origin of TGCTs can be traced back to primordial germ cells that fail to mature during embryonic development. These cells express high levels of pluripotency factors, including the transcription factor NANOG which is highly expressed in TGCTs. Gain or amplification of the NANOG locus is common in advanced tumours, suggesting a key role for this master regulator of pluripotency in TGCT stemness and malignancy. In this study, we analysed the expression of microRNAs (miRNAs) that are regulated by NANOG in TGCTs via integrated bioinformatic analyses of data from The Cancer Genome Atlas and NANOG chromatin immunoprecipitation in human embryonic stem cells. Through gain-of-function experiments, MIR9-2 was further investigated as a novel tumour suppressor regulated by NANOG. After transfection with MIR9-2 mimics, TGCT cells were analysed for cell proliferation, invasion, sensitivity to cisplatin, and gene expression signatures by RNA sequencing. For the first time, we identified 86 miRNAs regulated by NANOG in TGCTs. Among these, 37 miRNAs were differentially expressed in NANOG-high tumours, and they clustered TGCTs according to their subtypes. Binding of NANOG within 2kb upstream of the MIR9-2 locus was associated with a negative regulation. Low expression of MIR9-2 was associated with tumour progression and MIR9-2-5p was found to play a role in the control of tumour stemness. A gain of function of MIR9-2-5p was associated with reduced proliferation, invasion, and sensitivity to cisplatin in both embryonal carcinoma and seminoma tumours. MIR9-2-5p expression in TGCT cells significantly reduced the expression of genes regulating pluripotency and cell division, consistent with its functional effect on reducing cancer stemness. This study provides new molecular insights into the role of NANOG as a key determinant of pluripotency in TGCTs through the regulation of MIR9-2-5p, a novel epigenetic modulator of cancer stemness. Our data also highlight the potential negative feedback mediated by MIR9-2-5p on NANOG expression, which could be exploited as a therapeutic strategy for the treatment of TGCTs.

The T-locus - inspiration and distraction?

The T/t locus was a major focus of study by mouse geneticists during the 20th century. In the 70s, as the study of cell surface antigens controlling transplantation antigens was taking off, several laboratories hypothesized that alleles of this locus would control cell surface antigens important for embryonic development. One such antigen, the embryonal carcinoma F9 antigen was said to be an example. Other antigens were described on sperm and embryos that were said to be controlled by alleles at the T/t complex. These findings were later found to be false. The history of the findings and their refutation is described.

Abstract 2856: Multi-omics approaches for biomarker discovery and target therapy in pediatric germ cell tumors

Abstract Pediatric germ cell tumors (GCT) are rare and heterogeneous, presenting various histologies. There are no specific markers for diagnosing GCT, and 30% of patients are resistant to standard cisplatin-based chemotherapy, which can be related to epithelial-mesenchymal transition (EMT). Therefore, this study aims to elucidate markers for classifying GCTs, evaluate the molecular mechanisms related to drug resistance, and search for new therapeutic approaches. The expression of miRNAs was assessed for 42 GCT-pediatric patients through miRNA Panel - Nanostring technology. Whole Exome Sequencing (WES) was performed for 16 GCT-pediatric patients to assess somatic mutations. In silico analysis of epithelial-mesenchymal transition (EMT) markers was performed using the cBioPortal database. The expression of EMT markers was evaluated in vitro in parental and cisplatin-resistant models (NTERA-2R). Xenograft model-derived NTERA-2R was established and EMT markers were also assessed. We identified specific miRNA expression signatures for each histology, including 34 miRNAs for dysgerminomas, 13 for embryonal carcinomas, 25 for yolk sac tumors, and one for immature teratoma. We identified 26 miRNAs that were commonly expressed in malignant tumors, with six miRNAs (miR-302a-3p, miR-302b-3p, miR-371a-5p, miR-372-3p, miR-373-3p, miR-367-3p) showing significant overexpression. MiR-302b-3p had a significant association with all the evaluated clinical features. WES analysis showed that Single Base Substitution signature 39 was observed in 68.75% of samples and SBS22 in 12.5%. Copy number alterations were identified in 4, 7, 8, 10, 12, 21, and 22 chromosomes, with amplification of CDKN1B, KRAS, CCND2, ETV6, and KDM5A genes and deletions of KIT and PTEN genes. Somatic mutations in NYAP2, RHBDF2, MTOR, KIT, ATM, KRAS, and PIK3CA were frequent among the samples. KIT (Asp816Val, Ala829Pro) and KRAS (Gln61Leu) mutations were classified with potential clinical significance and were validated. In silico analysis showed that different histologies had distinct expression profiles for EMT markers. Patients with SNAILlowSLUGlow expression had higher PFS than those with SNAILhighSLUGhigh (p=0.006). The median PFS of patients with SLUGlow was 46.4 months, while SLUGhigh was 28.0 months (p=0.022). In vitro analysis using NTERA-2R showed overexpression of EMT markers. The same result was obtained in the xenograft model derived from NTERA-2R. Our study offers vital insights into pediatric GCTs' molecular landscape, using in silico, in vitro, in vivo analyses, and genetic assessments, highlighting three points: 1) The miRNA signatures for each histology may have robust diagnostic and prognostic potential, besides clinical implications as biomarkers. 2) KIT and KRAS genes are possible therapeutic targets of pediatric GCTs. 3) EMT is related to cisplatin resistance and SLUG is a potential molecular target. Citation Format: Ana Flavia Souza Peres, Ingridy Izabella Vieira Cardoso, Janaina Mello Soares Galvao, Ana Glenda Santarosa Vieira, Marcela Nunes Rosa, Isabela Cristiane Tosi, Daniel Antunes Moreno, Ana Carolina Laus, Andre van Helvoort Lengert, Silvia Aparecida Teixeira, Adriane Feijo Evangelista, Rui Manuel Reis, Luiz Fernando Lopes, Mariana Tomazini Pinto. Multi-omics approaches for biomarker discovery and target therapy in pediatric germ cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2856.

Prognostic factors for relapse in patients with clinical stage I testicular non-seminoma: A nationwide, population-based cohort study

BackgroundApproximately 30% of patients with clinical stage I non-seminoma (CSI-NS) relapse. Current risk stratification is based on lymphovascular invasion (LVI) alone. The extent to which additional tumor characteristics can improve risk prediction remains unclear. ObjectiveTo determine the most important prognostic factors for relapse in CSI-NS patients. Design, setting, and participantsPopulation-based cohort study including all patients with CSI-NS diagnosed in Denmark between 2013 and 2018 with follow-up until 2022. Patients were identified in the prospective Danish Testicular Cancer database. By linkage to the Danish National Pathology Registry, histological slides from the orchiectomy specimens were retrieved. Outcome measurements and statistical analysisHistological slides were reviewed blinded to the clinical outcome. Clinical data were obtained from medical records. The association between prespecified potential prognostic factors and relapse was assessed using Cox regression analysis. Model performance was evaluated by discrimination (Harrell’s C-index) and calibration. ResultsOf 453 patients included, 139 patients (30.6%) relapsed during a median follow-up of 6.3 years. Tumor invasion into the hilar soft tissue of the testicular hilum, tumor size, LVI and embryonal carcinoma were independent predictors of relapse. The estimated 5-year risk of relapse ranged from < 5% to > 85%, depending on the number of risk factors. After internal model validation, the model had an overall concordance statistic of 0.75. Model calibration was excellent. Conclusion and relevanceThe identified prognostic factors provide a much more accurate risk stratification than current clinical practice, potentially aiding clinical decision-making.

Component Patterns and Survival Outcomes in Patients with Mixed Malignant Ovarian Germ Cell Tumors: A Retrospective Cohort Study

AimsTo evaluate the component patterns and risk stratification in patients with mixed malignant ovarian germ cell tumors (mMOGCT). MethodsA retrospective study of 70 mMOGCT patients treated in our hospital between 2000 and 2022 was conducted. The recurrence-free survival (RFS), disease-specific survival (DSS), and risk stratification systems based on scoring the identified prognostic factors were assessed. ResultsYolk sac tumor component was the most common type (80%), followed by dysgerminoma (50%), immature teratoma (40%), embryonic carcinoma (27.1%), and chorionic carcinoma (15.7%). The 5-year RFS and DSS rates were 77.9% and 87.9%, respectively. International federation of gynecology and obstetrics (FIGO) stage III-IV (RR 3.253, P = 0.029) and normalization of tumor marker (TM) ≤ 3 cycles of chemotherapy (RR 6.249, P = 0.017) were risk factors for RFS and DSS, respectively. Significant DSS (RR 8.268, P = 0.006) was also noted between patients who had normalized TM ≤ 4 and ≥5 cycles of chemotherapy. FIGO stages I–II and stages III–IV were scored as 0 and 2, respectively. AFP normalization ≤3, 4, and ≥5 cycles of chemotherapy were scored as 0, 1, and 4, respectively. A total score of 0, 1–2, and ≥3 stratified patients into low-risk (43 patients), intermediate-risk (13 patients), and high-risk groups (14 patients), respectively. Patients in three risk stratifications manifested significant differences in DSS (P = 0.010) but not in RFS (P > 0.05). ConclusionDistinct different component patterns existed among mMOGCT patients, and predicting survival outcomes in a universal model was challenging.