Metastatic Testicular Germ Cell Tumors Research Articles

EHMT2/G9a and EZH2: Epimarkers in testicular germ cell tumors.

Testicular germ cell tumors remain the most frequent solid malignancies in young males. Despite excellent prognosis, the fact that only 60% of patients at diagnosis have elevated serum tumor markers (dependent on stage and histology) and the poor quality of life of patients who develop resistance to chemotherapy cannot be neglected. Consequently, it is mandatory to bring out novel biomarkers. The main goal was to evaluate EZH2 and EHMT2/G9a immunoexpression in a well-characterized patients' cohort of primary and metastatic testicular germ cell tumors, seeking associations with clinicopathological features and discovering differential immunoexpression patterns among specific subtypes. First, an in silico analysis of the Cancer Genome Atlas database was performed regarding EZH2 and EHMT2/G9a. Then, immunohistochemistry for EZH2 and EHMT2/G9a was carried out in a cohort of testicular germ cell tumor patients, comprising 155 chemo-naïve primary tumors and 11 chemo-treated metastases. Immunoexpression was evaluated using a digital pathology analysis software. Higher EZH2 and EHMT2/G9a expression levels were found in non-seminoma in the in silico analysis, particularly in embryonal carcinoma. Through digital pathology analysis, non-seminomas showed significantly higher EZH2 and EHMT2/G9a immunoexpression, with embryonal carcinoma showing higher expression. Moreover, mixed tumors with 50% or more of embryonal carcinoma component revealed the highest nuclei positivity for both biomarkers. Cisplatin-exposed metastases demonstrated a higher EZH2-positive nuclei and H-score, as well as higher EHMT2/G9a-positive nuclei. Overall, our data suggest that EZH2 and EHMT2/G9a might be associated with greater aggressiveness and, eventually, involved in the metastatic setting, paving the way for testing targeted therapies.

Identification of a panel of microRNA biomarkers for the management of patients with refractory germ cell tumor of the testis.

521 Background: In 1946, nearly 90% of patients diagnosed with metastatic Testicular Germ Cell Tumor (TGCT) succumbed to the disease within a year. In the present day, over 90% of such patients are successfully cured. However, there is a remaining 10% of patients have tumors that consist of chemotherapy-resistant forms of TGCT, such as yolk sac tumors and teratomas. This underlines the need for developing biomarkers that can guide the choice between additional chemotherapy and surgical intervention. MicroRNAs (miRNAs), non-coding RNA molecules that control post-transcriptional modifications, have shown promise in identifying TGCTs. Research has particularly highlighted the overexpression of miR-371a-3p and miR-375-3p in TGCTs. Nevertheless, these miRNAs are not effective in distinguishing between teratomas and other TGCT types. The Cancer Genome Atlas (TCGA), a publicly accessible database, offers clinical and miRNA data on various tumors, including TGCTs. Our objective is to leverage TCGA to identify miRNAs that can be used to classify TGCTs containing teratomas from those that do not. Methods: Between 2001 and 2013, TCGA gathered a total of 135 primary TGCT samples. We group these tumors into those without teratomas and those with any teratomatous components. We used EdgeR, a Bioconductor package designed for differential expression analysis of RNA that accounts for both biological and technical variation. A false discovery rate of less than 1e-8 was applied. A heatmap was generated to display the hierarchical clustering of differentially expressed miRNAs. Results: Based on the above cut-off, a panel of 50 miRNAs of differentially expressed miRNA were used to generate a hierarchical clustering that showed clear clustering between the two groups. Notably, tumor suppressor miRNAs such as hsa-mir-203a, hsa-mir-199, and hsa-mir-152, which are usually downregulated in non-teratoma TCGT, are overexpressed in teratomas. Other miRNA including hsa-mir-146a and hsa-mir-3607, which are downregulated tumor suppressors for non-TCGT are also downregulated in teratomas but not in teratoma-free TGCT. Conclusions: Despite the high cure rate for TGCTs, tumors containing chemotherapy-resistant teratomas still contribute to elevated mortality rates. Utilizing TCGA data, we identified a set of miRNAs that are effective in distinguishing between teratoma-containing and teratoma-free TGCTs. This miRNA panel has the potential to inform decisions related to surgical treatment plans and provide further insight into the biology of the disease.

SMARCB1-Deficient Skull Base Chondrosarcoma with 12p Duplication Presenting as Somatic-Type Malignancy Arising from Metastatic Seminoma

Somatic-type malignancy (STM) can occur infrequently within a primary or metastatic testicular germ cell tumor (TGCT) and is associated with dismal prognosis and survival. STM with chondrosarcomatous features is exceedingly rare and head and neck involvement has not been previously documented. A 39-year-old white man presented with nasal obstruction and epistaxis. Imaging disclosed a 6.9-cm expansile tumor involving the nasal cavity and skull base with intraorbital and intracranial extension. The histopathologic properties of the tumor were compatible with chondrosarcoma, grade II-III. Immunohistochemically, malignant cells were strongly and diffusely positive for S100 and epithelial markers, and showed loss of SMARCB1 expression. IDH1/2 mutations were not detected. Following whole-body PET scan, a 7.0-cm left testicular mass was discovered and diagnosed as seminoma with syncytiotrophoblastic cells, stage pT3NXM1b. Extensive retroperitoneal, mediastinal, and supraclavicular lymphadenopathy was also noticed. Histopathologic examination of the left supraclavicular lymph node revealed metastatic seminoma. By FISH, most metastatic nodal seminoma cells harbored 1 to 4 copies of isochromosome 12p, while the chondrosarcoma featured duplication of 12p. Presence of a malignant TGCT with disseminated supradiaphragmatic lymphadenopathy, the unique immunophenotypic properties of the skull-based chondrosarcoma and lack of IDH1/2 aberrations with gain of 12p strongly support the diagnosis of STM chondrosarcoma arising from metastatic TGCT. The patient did not respond to chemotherapy and succumbed three months after diagnosis. Although exceedingly uncommon, metastasis to the head and neck may occur in patients with TGCT. This case of STM chondrosarcoma demonstrated divergent immunophenotypic and molecular characteristics compared to “typical” examples of head and neck chondrosarcoma. High index of suspicion is advised regarding the diagnosis of lesions that present with otherwise typical histomorphology but unexpected immunohistochemical or molecular features.

Simultaneous bilateral pulmonary and cardiac invasion of metastatic testicular germ cell tumor: A rare case report

AbstractA 23‐year‐old man with a history of a testicular germ cell tumor (GCT) that was incompletely treated, is presented with dyspnea. Significant bilateral pulmonary masses were seen on computed tomography (CT). A large, hypermobile mass in the left atrium (LA) as well as mitral valve dysfunction and moderate pulmonary hypertension was detected by transthoracic echocardiography (TTE). Urgent cardiac surgery was performed to prevent the tumor embolism in addition to restore mitral valve function and reduce pulmonary hypertension. Pathologic reports confirmed testicular GCT. GCT metastasis to the heart and lungs is an incredibly rare phenomenon. To our knowledge, this is the first case of simultaneous GCT metastasis to both lungs and LA. This article highlights the importance of cardiac examinations and imaging in germ cell tumor patients. If there is a functional complication for vital organs such as the heart or lungs, surgical interventions are given priority before starting chemotherapy.

Biomarkers for Salvage Therapy in Testicular Germ Cell Tumors

The outcome of metastatic testicular germ cell tumor patients has been dramatically improved by cisplatin-based chemotherapy combinations. However, up to 30% of patients with advanced disease relapse after first-line therapy and require salvage regimens, which include treatments with conventional-dose chemotherapy or high-dose chemotherapy with autologous stem cell transplantation. For these patients, prognosis estimation represents an essential step in the choice of medical treatment but still remains a complex challenge. The available histological, clinical, and biochemical parameters attempt to define the prognosis, but they do not reflect the tumor’s molecular and pathological features and do not predict who will exhibit resistance to the several treatments. Molecular selection of patients and validated biomarkers are highly needed in order to improve current risk stratification and identify novel therapeutic approaches for patients with recurrent disease. Biomolecular biomarkers, including microRNAs, gene expression profiles, and immune-related biomarkers are currently under investigation in testicular germ cell tumors and could potentially hold a prominent place in the future treatment selection and prognostication of these tumors. The aim of this review is to summarize current scientific data regarding prognostic and predictive biomarkers for salvage therapy in testicular germ cell tumors.

Evaluation of a miRNA-371a-3p Assay for Predicting Final Histopathology in Patients Undergoing Primary Nerve-sparing Retroperitoneal Lymphadenectomy for Stage IIA/B Seminoma or Nonseminoma

Patients with marker-negative clinical stage IIA/B seminoma or nonseminoma represent a therapeutic challenge, as 20–30% might harbor nonmalignant histologies. MicroRNA 371a-3p (miR371) may represent a biomarker with diagnostic and predictive properties in testicular germ cell tumors (TGCTs). We evaluated the predictive accuracy of this biomarker in identifying the presence or absence of lymph node metastases (LNMs) in clinical stage IIA/B TGCT. In a cohort of 24 consecutive patients with marker-negative clinical stage IIA/B TGCT (n = 15 seminoma, n = 9 nonseminoma) serum miR371 was assessed 1 d before nerve-sparing retroperitoneal lymphadenectomy. Histology revealed metastatic TGCT in 22/24 patients (91.7%), with positive miR371a findings for 20 of these 22 patients with metastases (90.9%). Histology revealed no malignancy in one patient and lymphoma in another, both of whom had negative miR371a findings. One additional patient with pure teratoma and one with a microscopic seminomatous LNM had false-negative miR371a findings. The miR371 assay had sensitivity of 90.9% and specificity of 50%. The positive predictive value was 100.0% and the negative predictive value was 75.0%. According to the data available, miR371a represents a highly reliable, personalized tumor marker for predicting the presence of low-volume retroperitoneal LNMs in marker-negative TGCT. miR371 has potential for inclusion in the diagnostic armamentarium for men with equivocal lymph nodes to facilitate avoidance of unnecessary treatment and the associated toxicity. Patient summaryOur study demonstrates that blood tests for the biomarker miR371 are highly reliable in predicting the presence of lymph node metastases in patients with stage IIA/B testicular cancer. For patients with equivocal findings, use of this test may help in avoiding unnecessary treatment.

Strong apoptotic response of testis tumor cells following cisplatin treatment

Most solid metastatic cancers are resistant to chemotherapy. However, metastatic testicular germ cell tumors (TGCT) are cured in over 80% of patients using cisplatin-based combination therapy. Published data suggest that TGCTs are sensitive to cisplatin due to limited DNA repair and presumably also to a propensity to undergo apoptosis. To further investigate this aspect, cisplatin-induced activation of apoptotic pathways was investigated in cisplatin-sensitive testis tumor cells (TTC) and compared to cisplatin-resistant bladder cancer cells. Apoptosis induction was investigated using flow cytometry, caspase activation and PARP-1 cleavage. Immunoblotting and RT-PCR were applied to investigate pro- and anti-apoptotic proteins. Transfections were performed to target p53- and Fas/FasL-mediated apoptotic signaling. Immunoblotting experiments revealed p53 to be induced in TTC, but not bladder cancer cells following cisplatin. Higher levels of pro-apoptotic Bax and Noxa were observed in TTC, anti-apoptotic Bcl-2 was solely expressed in bladder cancer cells. Cisplatin led to translocation of Bax to the mitochondrial membrane in TTC, resulting in cytochrome C release. Cisplatin increased the expression of FasR mRNA and FasL protein in all tumor cell lines. Targeting the apoptotic pathway via siRNA-mediated knockdown of p53 and FAS reduced death receptor-mediated apoptosis and increased cisplatin resistance in TTC, indicating the involvement of FAS-mediated apoptosis in the cisplatin TTC response. In conclusion, both the death receptor and the mitochondrial apoptotic pathway become strongly activated in TTC following cisplatin treatment, explaining, together with attenuated DNA repair, their unique sensitivity toward platinum-based anticancer drugs.

Radiomics Analyses to Predict Histopathology in Patients with Metastatic Testicular Germ Cell Tumors before Post-Chemotherapy Retroperitoneal Lymph Node Dissection.

The identification of histopathology in metastatic non-seminomatous testicular germ cell tumors (TGCT) before post-chemotherapy retroperitoneal lymph node dissection (PC-RPLND) holds significant potential to reduce treatment-related morbidity in young patients, addressing an important survivorship concern. To explore this possibility, we conducted a study investigating the role of computed tomography (CT) radiomics models that integrate clinical predictors, enabling personalized prediction of histopathology in metastatic non-seminomatous TGCT patients prior to PC-RPLND. In this retrospective study, we included a cohort of 122 patients. Using dedicated radiomics software, we segmented the targets and extracted quantitative features from the CT images. Subsequently, we employed feature selection techniques and developed radiomics-based machine learning models to predict histological subtypes. To ensure the robustness of our procedure, we implemented a 5-fold cross-validation approach. When evaluating the models' performance, we measured metrics such as the area under the receiver operating characteristic curve (AUC), sensitivity, specificity, precision, and F-score. Our radiomics model based on the Support Vector Machine achieved an optimal average AUC of 0.945. The presented CT-based radiomics model can potentially serve as a non-invasive tool to predict histopathological outcomes, differentiating among fibrosis/necrosis, teratoma, and viable tumor in metastatic non-seminomatous TGCT before PC-RPLND. It has the potential to be considered a promising tool to mitigate the risk of over- or under-treatment in young patients, although multi-center validation is critical to confirm the clinical utility of the proposed radiomics workflow.

Long-term response to camrelizumab in a pretreated metastatic mixed testicular germ-cell tumor patient with co-mutations in DNA damage-repair genes.

Little information is available regarding the therapeutic efficacy of immune checkpoint inhibitors and the prediction of DNA damage-repair (DDR) genes in mixed testicular germ-cell tumors (TGCTs). Here we report a pretreated patient with metastatic mixed TGCT harboring variations of three important DDR genes - BRCA2, MSH6 and PMS2 - identified by next-generation sequencing using plasma-based circulating tumor DNA. He obtained stable clinical benefit from PD-1 blockade. At the latest follow-up, he had a progression-free survival of more than 28 months and had survived 6.75 years since diagnosis. To our knowledge, this case is the first report of long-term clinical outcome obtained from immune checkpoint inhibitor therapy in a pretreated patient with mixed metastatic TGCT harboring co-mutations in DDR genes.

Multimodal treatment of testicular cancer: chemotherapy, surgery or radiotherapy?

Metastatic testicular germ cell tumors patients require histology- and stage-appropriate therapy to achieve optimal therapeutic outcomes. This work focuses on the interdisciplinary presentation of current recommendations for the treatment of metastatic germ cell tumor patients. The interdisciplinary recommendations were formulated based on the German S3guideline and supplemented by recent literature. Using astage-specific and guideline-based treatment approach, interdisciplinary cooperation between urology, oncology, and radiotherapy is mandatory to successfully achieve ahigh rate of cure and, in the case of complex advanced tumors, also the most effective therapy possible. The question of optimal treatment approaches for seminoma in cSIIA/B remains particularly challenging. Since treatment of advanced or multiple relapsed germ cell tumor patients remains complex, patients should be referred for an online second opinion ( https://urologie.ekonsil.org ).

Post-treatment Residual Clinicopathological Outcomes in Testicular Germ Cell Tumours.

Surgical resection is a generally accepted treatment for residual masses after chemotherapy for metastatic testicular germ cell tumour (GCT). About half the patients have necrosis in post-chemotherapy residual masses, whereas rest have viable tumour and teratoma. The likelihood of leaving behind teratoma with its subsequent complications such as growing teratoma syndrome necessitates resection outweighing its surgical complications. Ours is a retrospective observational study and aims at assessing post-chemotherapy residual masses in testicular GCTs and to predict importance of teratomatous and non-seminomatous components. A total of 62 cases of testicular GCTs resected after chemotherapy between January 2012 and June 2019 were included. Demographic, clinical, biochemical and imageological findings were noted and categorised according to WHO classification (2016). They were divided into two groups - those who underwent retroperitoneal lymph node dissection (RPLND) post-high inguinal orchidectomy (HIO) and chemotherapy (CT) as group 1 (n = 40) and those who underwent HIO and/or RPLND post-chemotherapy as group 2 (n = 22). The gross and microscopic examination was carried out to assess response to chemotherapy in terms of residual viable tumour, necrosis and teratoma. Viable tumour, necrosis and teratoma were 10%, 62.5% and 35% respectively in group 1 and in group 2, the same were 15%, 70% and 25% respectively in HIO specimen and 7%, 50% and 21% respectively in RPLND specimen. All the cases with viable tumour were proven to be yolk sac tumours (YST) based on morphology and immunohistochemistry (IHC).Twenty cases had teratoma in the post-CT residual masses out of which 11 cases had teratoma despite reduction in size. At a median follow-up of 47.85months, 5 cases in group 1 and 2 cases in group 2 showed relapse and it was observed that group 1 had a prolonged relapse-free survival over group 2. Our study re-emphasises the importance of performing resection of residual mass post-CT irrespective of the size, imageological or biochemical evidence of tumour regression. There does not appear to be reliable predictors of post-chemotherapy histology of residual masses indicating the continued need for surgical resection in specialised centres.

Iso-chromosome 12p Analysis by Fluorescent In-Situ Hybridization: An Academic Institutional Experience

Abstract Introduction/Objective Chromosome 12 abnormalities like iso-chromosome 12p (i12p) and amplification of 12p are seen in majority (89%) of the primary and metastatic testicular germ cell tumors (TGCTs). i12p can be detected by karyotyping, fluorescent in-situ hybridization (FISH) or reverse transcriptase polymerase chain reaction. The aim of this study was to review i12p FISH data at our institution and assess the clinical utility. Methods/Case Report Laboratory information system was queried over a period of 15 years to search for cases where i12p FISH test was requested. FISH test was performed using TelVysion 12p telomeric probe and CEP 12 centromere probe on paraffin-embedded tissue or cell blocks. A ratio of 12ptel/CEP12 signal of 1.4 or greater was considered as positive. Patient demographics, clinical presentation, pathologic findings, and follow-up data were documented and correlated. Results (if a Case Study enter NA) Total 58 cases were identified with an age range of 14 to 76 years. Majority were male (M=52, F=6). Of these cases, 15 were testicular and 43 extra-testicular cases that included resection (n=35), biopsy (n=20) and cell-blocks (n=3). i12p was detected in 8 out of 15 testicular cases while i12p was detected in 16 out of the 43 extra-testicular cases. The extra- testicular cases included 17 retroperitoneal lesions, 8 lesions from the mediastinum, 6 lymph nodes from other sites and 12 miscellaneous lesions. Using pathology diagnosis with immunohistochemistry as gold standard, overall sensitivity was 60% and specificity was 86%. There were 3 false positive cases [Benign testicular parenchyma (n=1), suspicious for germ cell neoplasia in-situ (n=1) and undifferentiated epithelioid neoplasm (n=1)]. Conclusion Our results show that although the sensitivity was limited, FISH test for i12p demonstrated high specificity(86%) for diagnosis of primary or metastatic TGCTs. As an adjunct test, i12p FISH can help identify and further characterize a significant number of GCTs with unusual morphology or clinical presentation.

Burned-Out Testicular Tumors in Adolescents: Clinical Aspects and Outcome.

Purpose: Testicular germ cell tumors are the fourth most common neoplasm in adolescents, accounting for 8% of all tumors in the age group 15–19 years. On rare instances, the primary testicular lesion is not clinically or radiologically evident while nodal or visceral metastases represent the clinical manifestations of the disease. This phenomenon is described as “burned-out testicular tumor.” In this paper, the authors report a single-institution experience with burned-out testicular tumors in adolescents and discuss their clinical implications.Patients and Methods: All the patients diagnosed with metastatic testicular germ cell tumors at Bambino Gesù Children Hospital between January 1, 2010, and June 30, 2020, were included in the study. Patients were categorized into two groups: “primary testicular” and “burned out.” All the patients were staged and treated according to the AIEOP–TCGM 2004 protocol.Results: Eleven patients were classified as “primary testicular,” and five patients were classified as “burned out.” “Burned-out” tumors were associated with the presence of systemic symptoms compared to “primary testicular” tumors (80 vs. 0%; p = 0.0027) and higher aFP, hCG, and LDH levels (p < 0.00001). The “burned-out” population had a statistically significant higher incidence of relevant toxicity than the “primary testicular” population (80 vs. 18%; p = 0.0357) and a worse outcome in terms of both mean overall survival (15 vs. 43 months; p = 0.0299) and mean event-free survival (12 vs. 38 months; p = 0.0164).Conclusion: “Burned-out” testicular tumors seem to be a well-distinct clinical entity with a high treatment-related toxicity and poor prognosis. Further studies are needed to clarify the “burned-out phenomenon” and to identify more effective therapeutic strategies for these patients.

Indications, feasibility and outcome of robotic retroperitoneal lymph node dissection for metastatic testicular germ cell tumours

Data on robotic retroperitoneal lymph node dissection (R-RPLND) for metastatic testicular germ cell tumours (mTGCTs) are scarce and the use of R-RPLND itself is still under debate. The aim of our study was to evaluate the indications, feasibility and outcomes of R-RPLND, with special emphasis on differences between primary R-RPLND (pR-RPLND) and post-chemotherapeutic R-RPLND (pcR-RPLND) in mTGCTs. We retrospectively analysed the data of patients who underwent R-RPLND for mTGCT between November 2013 and September 2019 in two centres in Germany. Indications, operative technique, intra- and postoperative complications and oncologic outcome were analysed. Twenty-three mTGCT patients underwent R-RPLND (7 pR-RPLND, 16 pcR-RPLND). For pR-RPLND versus pcR-RPLND, median time of surgery was 243 min [interquartile range (IQR) 123–303] versus 359 min (IQR 202–440, p = 0.154) and median blood loss 100 mL (IQR 50–200) versus 275 mL (IQR 100–775, p = 0.018). Intra- and postoperative complications were more frequent in pcR-RPLND (pcR-RPLND: intra/post: 44%/44%; pR-RPLND: intra/post: 0%/29%). However, these were only statistically significant in the case of intraoperative complications (intra: p = 0.036, post: p = 0.579). Intraoperative complications (n = 7), conversions (n = 4) and transfusions (n = 4) occurred in pcR-RPLND patients only. After a median follow-up of 16.3 months (IQR 7.5–35.0) there were no recurrences or deaths. R-RPLND displays a valuable, minimally invasive treatment option in mTGCT. However, R-RPLND is challenging and pcR-RPLND especially bears a considerable risk of complications. This operation should be limited to patients with an easily accessible residual tumour mass and to surgeons experienced in robotic surgery and TGCT treatment.

Differential methylation EPIC analysis discloses cisplatin-resistance related hypermethylation and tumor-specific heterogeneity within matched primary and metastatic testicular germ cell tumor patient tissue samples

Testicular germ cell tumors (TGCTs) are among the most common solid malignancies in young-adult men, and currently most mortality is due to metastatic disease and emergence of resistance to cisplatin. There is some evidence that increased methylation is one mechanism behind this resistance, stemming from individual studies, but approaches based on matched primary and metastatic patient samples are lacking. Herein, we provide an EPIC array-based study of matched primary and metastatic TGCT samples. Histology was the major determinant of overall methylation pattern, but some clustering of samples related to response to cisplatin was observed. Further differential analysis of patients with the same histological subtype (embryonal carcinoma) disclosed a remarkable increase in net methylation levels (at both promoter and CpG site level) in the patient with cisplatin-resistant disease and poor outcome compared to the patient with complete response to chemotherapy. This further confirms the recent results of another study performed on isogenic clones of sensitive and resistant TGCT cell lines. Differentially methylated promoters among groups of samples were mostly not shared, disclosing heterogeneity in patient tissue samples. Finally, gene ontology analysis of cisplatin-resistant samples indicated enrichment of differentially hypermethylated promoters on pathways related to regulation of immune microenvironment, and enrichment of differentially hypomethylated promoters on pathways related to DNA/chromatin binding and regulation. This data supports not only the use of hypomethylating agents for targeting cisplatin-resistant disease, but also their use in combination with immunotherapies and chromatin remodelers.

Expression of programmed death ligand-1 (PD-L1) in metastatic and postchemotherapy viable testicular germ cell tumors

IntroductionChemotherapy for testicular germ cell tumors (GCT) is highly effective, with few patients who do not respond. Clinical studies to evaluated novel treatments are challenging given the rarity of these patients. Therefore, we sought to evaluate PD-L1 staining on metastatic and postchemotherapy viable testicular GCTs as a surrogate for potential benefit for immunotherapy targeting the PD-1/PD-L1 axis. MethodsEthics research committee approval for this retrospective study was obtained by four participating institutions (CHU de Québec, St. Joseph's Health Care, Halifax Health Science Centre, Johannes Gutenberg University). Patients with viable metastatic testicular GCTs pathology samples were included. Patients with pure teratoma were excluded. PD-L1 staining with the 22C3 clone was evaluated on samples with >100 viable tumor cells using the combined positive score (CPS). ResultsFrom 51 patients identified at participating institutions, 24 postchemotherapy and 18 chemotherapy-naive metastatic samples were available for PD-L1 staining, with 9 matched prechemotherapy samples and 7 matched orchiectomy pathology samples, respectively. The median CPS score was 55.6 (IQR 16–100) for all metastatic samples, 44.9 (IQR 13–100) for postchemotherapy metastatic samples, and 68.8 (IQR 38–100) for chemotherapy-naïve metastatic samples, with the median number of viable tumor cells at 545, 500, and 550, respectively. Differences were not significant between chemotherapy-naïve and postchemotherapy samples (P = 0.07), though among non-seminoma GCT metastatic samples, CPS scores were significantly lower postchemotherapy (P = 0.02). Significant differences among postchemotherapy metastatic tumors were also seen according to predominant subtype, with lower CPS scores for predominant yolk sac and higher values for predominant seminoma and choriocarcinoma. In 7 patients with matched specimens pre- and postchemotherapy, a significant increase in CPS was observed for seminoma (26.7 vs. 81.7, P = 0.045), but not nonseminoma GCTs. Comparing all chemotherapy naïve-samples, PD-L1 expression was higher in metastatic samples versus testicular samples (mean CPS 68.8 vs. 39.8, P = 0.02). This was also seen in matched chemotherapy-naïve samples (mean CPS 77.9 vs. 33.1, P = 0.01). ConclusionOur results suggest that most patients with refractory GCTs postchemotherapy will not benefit from PD-1/PD-L1 immunotherapy. However, the high PD-L1 expression in patients with predominant or pure seminoma post-chemotherapy suggests this may represent a subgroup for whom further trials may be considered.

P0713 / #1204: HEPATIC SINUSOIDAL OBSTRUCTION SYNDROME AS A CAUSE OF MULTIORGAN DYSFUNCTION IN TWO PEDIATRIC ONCOLOGY PATIENTS WITH FIRST-LINE CHEMOTHERAPY

Aims & Objectives: Hepatic Sinusoidal obstruction syndrome (SOS/VOD) is a toxic complication common after stem cell transplant (HSCT). It may also occur after chemotherapy without HSCT. We present two cases who developed severe SOS/VOD with multiorgan dysfunction syndrome (MODS) after chemotherapy. Methods: Retrospective case and literature review. Results: Patient 1 was 3-month-old male with infantile fibrosarcoma. He received actinomycin and vincristine two days prior. He was admitted to PICU for sepsis quickly developing hepatic-predominant (Total Bilirubin 4.6 TGO 6457 TGP 1838, ascites) MODS. Infectious etiologis including bacteremia, CNS infection and cytomegalovirus were excluded. Doppler ultrasound revealed portal hypertension with reverse flow. Biopsy showed sinusoidal dilation and congestion. Patient received methylprednisolone pulses (500mgm2do every q12, 6 doses). Hepatic dysfunction improved but course was complicated with Candidemia and intestinal perforation. Patient ultimately recovered and was discharged from PICU after 72 days. Patient 2 was 17-year-old male with metastatic testicular germ-cell tumor. He received bleomycin, carboplatin and etoposide 5 days prior. He was admitted to PICU for shock and developed hepatic-predominant (total bilirubin 9, direct 7.1, ascites) MODS. Infectious causes were excluded. Doppler ultrasound revealed portal hypertension and reverse flow. He received methylprednisolone pulses (500mgm2do every 12 hours) interrupted at 3 doses because of developing pancreatitis, with fluconazole prophylaxis. Patient recovered and was discharged from PICU after 21 days Conclusions: SOS/VOD is a rare but severe cause of MODS in children with cancer. In cases where defibrotide is not available, methylprednisolone may be a therapeutic option albeit with important adverse events.

The decisive role of molecular pathology in presumed somatic metastases of type II testicular germ cell tumors: report of 2 cases

BackgroundMolecular diagnostics can be decisive in the differential diagnosis between a somatic metastasis of type II testicular germ cell tumor (TGCT) or a second primary carcinoma. This is in line with recent recommendations from the International Society of Urological Pathology, based on an international survey which showed that molecular testing is currently only performed by a minority of urological pathologists.Case presentationsThis case report illustrates the necessity of molecular testing in two patients with a history of type II TGCT and a metastatic (retro) peritoneal carcinoma years later. The genetic hallmark of type II TGCT, chromosome 12p gain, was studied by fluorescence in situ hybridization and whole genome methylation profiling in case 1, and by single nucleotide polymorphism (SNP)-array in case 2. Next generation sequencing (NGS) was used to further explore clonality between the primary TGCT and peritoneal metastasis in case 2. In case 1, chromosome 12p gain was found in the primary type II TGCT and in the acinar cell carcinoma of the metastatic malignancy. In case 2, SNP array showed 12p gain in the epithelial component of the primary teratomatous TGCT but not in the peritoneal adenocarcinoma. Furthermore, NGS showed no mutations in the primary teratomatous TGCT but a KRAS and GNAS mutation in the peritoneal adenocarcinoma, suggestive of an appendicular origin.ConclusionsWithout the molecular data, both cases would have been regarded as a metastatic TGCT with development of somatic-type malignancy, which appeared a wrong diagnosis for case 2. These cases demonstrate the importance of molecular methods as an adjunct in today’s pathology practice.

WNT signalling in the normal human adult testis and in male germ cell neoplasms.

Is WNT signalling functional in normal and/or neoplastic human male germ cells? Regulated WNT signalling component synthesis in human testes indicates that WNT pathway function changes during normal spermatogenesis and is active in testicular germ cell tumours (TGCTs), and that WNT pathway blockade may restrict seminoma growth and migration. Regulated WNT signalling governs many developmental processes, including those affecting male fertility during early germ cell development at embryonic and adult (spermatogonial) ages in mice. In addition, although many cancers arise from WNT signalling alterations, the functional relevance and WNT pathway components in TGCT, including germ cell neoplasia in situ (GCNIS), are unknown. The cellular distribution of transcripts and proteins in WNT signalling pathways was assessed in fixed human testis sections with normal spermatogenesis, GCNIS and seminoma (2-16 individuals per condition). Short-term (1-7 h) ligand activation and long-term (1-5 days) functional outcomes were examined using the well-characterised seminoma cell line, TCam-2. Pathway inhibition used siRNA or chemical exposures over 5 days to assess survival and migration. The cellular localisation of WNT signalling components was determined using in situ hybridisation and immunohistochemistry on Bouin's- and formalin-fixed human testis sections with complete spermatogenesis or germ cell neoplasia, and was also assessed in TCam-2 cells. Pathway function tests included exposure of TCam-2 cells to ligands, small molecules and siRNAs. Outcomes were measured by monitoring beta-catenin (CTNNB1) intracellular localisation, cell counting and gap closure measurements. Detection of nuclear-localised beta-catenin (CTNNB1), and key WNT signalling components (including WNT3A, AXIN2, TCF7L1 and TCF7L2) indicate dynamic and cell-specific pathway activity in the adult human testis. Their presence in germ cell neoplasia and functional analyses in TCam-2 cells indicate roles for active canonical WNT signalling in TGCT relating to viability and migration. All data were analysed to determine statistical significance. No large-scale datasets were generated in this study. As TGCTs are rare and morphologically heterogeneous, functional studies in primary cancer cells were not performed. Functional analysis was performed with the only well-characterised, widely accepted seminoma-derived cell line. This study demonstrated the potential sites and involvement of the WNT pathway in human spermatogenesis, revealing similarities with murine testis that suggest the potential for functional conservation during normal spermatogenesis. Evidence that inhibition of canonical WNT signalling leads to loss of viability and migratory activity in seminoma cells suggests that potential treatments using small molecule or siRNA inhibitors may be suitable for patients with metastatic TGCTs. This study was funded by National Health and Medical Research Council of Australia (Project ID 1011340 to K.L.L. and H.E.A., and Fellowship ID 1079646 to K.L.L.) and supported by the Victorian Government's Operational Infrastructure Support Program. None of the authors have any competing interests.

'Snail factors in testicular germ cell tumours and their regulation by the BMP4 signalling pathway'.

Snail transcription factors mediate key cellular transitions in many developmental processes, including spermatogenesis, and their production can be regulated by TGF-β superfamily signalling. SNAI1 and SNAI2 support many cancers of epithelial origin. Their functional relevance and potential regulation by TGF-β superfamily ligands in germ cell neoplasia are unknown. SNAI1, SNAI2 and importin 5 (IPO5; nuclear transporter that selectively mediates BMP signalling) cellular localization was examined in fixed normal adult human and/or neoplastic testes using in situ hybridization and/or immunohistochemistry. SNAI1 and SNAI2 functions were assessed using the well-characterized human seminoma cell line, TCam-2. Cell migration, adhesion/proliferation and survival were measured by scratch assay, xCELLigence and flow cytometry following siRNA-induced reduction of SNAI1 and SNAI2 in TCam-2 cells. The potential regulation of SNAI1 and SNAI2 in TCam-2 cells by TGF-β signalling ligands, activin A and BMP4 was evaluated following 48hours culture, including with siRNA regulation of IPO5 to selectively restrict BMP4 signalling. In normal testes, SNAI1 transcript was identified in some spermatogonia and in spermatocytes, and SNAI2 protein localized to nuclei of spermatogonia, spermatocytes and round spermatids. In neoplastic testes, both SNAI1 and SNAI2 were detected in GCNIS and in seminoma cells. SNAI1 and SNAI2 reduction in TCam-2 cells by siRNAs significantly inhibited migration and survival, respectively. Exposure to BMP4, but not activin A, significantly increased SNAI2 (~18-fold). IPO5 inhibition by siRNAs decreased BMP4-induced SNAI2 upregulation (~5-fold). Additionally, SNAI2 reduction using siRNAs inhibited BMP4-induced TCam-2 cell survival. This is the first evidence that SNAI1 and SNAI2 are involved in human spermatogenesis, with independent functions. These outcomes demonstrate that SNAI1 and SNAI2 inhibition leads to loss of migratory and viability capacities in seminoma cells. These findings show the potential for therapeutic treatments targeting SNAIL or BMP4 signalling for patients with metastatic testicular germ cell tumours.