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Testicular cancer is the most common cancer type among young men. Despite highly effective cisplatin-based chemotherapy, around 20% of patients with metastatic disease will still die from the disease. The aim of this study was to explore the use of kinase inhibitors to sensitize testicular cancer cells to cisplatin treatment. Activation of kinases, including receptor tyrosine kinases and downstream substrates, was studied in five cisplatin-sensitive or -resistant testicular cancer cell lines using phospho-kinase arrays and Western blotting. The phospho-kinase array showed AKT and S6 to be among the top phosphorylated proteins in testicular cancer cells, which are part of the PI3K/AKT/mTORC pathway. Inhibitors of most active kinases in the PI3K/AKT/mTORC pathway were tested using apoptosis assays and survival assays. Two mTORC1/2 inhibitors, AZD8055 and MLN0128, strongly enhanced cisplatin-induced apoptosis in all tested testicular cancer cell lines. Inhibition of mTORC1/2 blocked phosphorylation of the mTORC downstream proteins S6 and 4E-BP1. Combined treatment with AZD8055 and cisplatin led to reduced clonogenic survival of testicular cancer cells. Two testicular cancer patient-derived xenografts (PDX), either from a chemosensitive or -resistant patient, were treated with cisplatin in the absence or presence of kinase inhibitor. Combined AZD8055 and cisplatin treatment resulted in effective mTORC1/2 inhibition, increased caspase-3 activity, and enhanced tumor growth inhibition. In conclusion, we identified mTORC1/2 inhibition as an effective strategy to sensitize testicular cancer cell lines and PDX models to cisplatin treatment. Our results warrant further investigation of this combination therapy in the treatment of patients with testicular cancer with high-risk relapsed or refractory disease.

mTOR knock down in combination with cisplatin in resistant TC cells

PDGFRbeta protein expression in TC cell lines

Cisplatin sensitivity in TC cells and RTK validation on western blot

mTORC1/2 downstream pathway evaluation in Tera cells

Autophagy induction and the effect on apoptosis in cisplatin resistant TC cells

mTORC1/2 inhibition in combination with cisplatin in TC PDX models

Dasatinib in combination with cisplatin in resistant TC cells

Supplementary Data from Mapatumumab, a Fully Human Agonistic Monoclonal Antibody That Targets TRAIL-R1, in Combination with Gemcitabine and Cisplatin: a Phase I Study

Supplementary methods, Supplementary Fig. S1, Supplemtary Table S1, Supplementary Table S2, Supplementary Table S3

A. PET uptake in the circulation per cohort, as determined by region of interest drawn in left ventricle; cohort 1 (in blue) contained 2 patients (0 mg additional MMOT0530A), cohort 2 (in green) contained 9 patients (10 mg additional MMOT0530A). On X axis the different PET moments: 2, 4 and 7 days post injection. On the Y axis the mean SUVmax per cohort.

Supplementary Tables

Purpose: To evaluate the safety, tolerability, pharmacokinetics, and antitumor activity of mapatumumab, a fully human monoclonal antibody targeting tumor necrosis factor–related apoptosis–inducing ligand receptor 1 (TRAIL-R1), in combination with gemcitabine and cisplatin.Experimental Design: Patients with advanced solid tumors received gemcitabine 1,250 mg/m2 i.v. on days 1 and 8 and cisplatin 80 mg/m2 i.v. on day 1 of each 21-day cycle. Escalating mapatumumab doses were administered i.v. every 21 days. Toxicity was evaluated and pharmacokinetic analysis of plasma mapatumumab, gemcitabine, 2-difluoro-2-deoxyuridine, and unbound and total platinum was done. TRAIL-R1 tumor expression was determined immunohistochemically.Results: Forty-nine patients received mapatumumab (1 mg/kg, n = 4; 3 mg/kg, n = 7; 10 mg/kg, n = 12; 20 mg/kg, n = 13; or 30 mg/kg, n = 13). A median of six cycles (range, 1-48) was administered. The adverse events most commonly observed reflect the toxicity profile of gemcitabine and cisplatin. Dose-limiting toxicities were seen in 3 of 12 patients at 10 mg/kg, consisting of grade 3 transaminitis, neutropenic fever, and grade 4 thrombocytopenia. At 20 mg/kg, 2 of 12 patients had dose-limiting toxicities, including grade 4 thrombocytopenia and grade 4 fatigue. The maximum tolerated dose was not reached. Pharmacokinetic interactions have not been observed. Twelve patients had a partial response, and 25 patients showed stable disease with a median duration of 6 months.Conclusions: Mapatumumab in combination with gemcitabine and cisplatin is safe and well tolerated at doses up to 30 mg/kg. Further studies on this combination are warranted. (Clin Cancer Res 2009;15(17):5584–90)

Purpose:Biodistribution of bispecific antibodies in patients is largely unknown. We therefore performed a feasibility study in 9 patients with advanced gastrointestinal adenocarcinomas to explore AMG 211 biodistribution (also known as MEDI-565), an approximately 55 kDa bispecific T-cell engager (BiTE®) directed against carcinoembryonic antigen (CEA) on tumor cells and cluster of differentiation 3 (CD3) on T-cells.Experimental Design:89Zr-labeled AMG 211 as tracer was administered alone or with cold AMG 211, for PET imaging before and/or during AMG 211 treatment.Results:Before AMG 211 treatment, the optimal imaging dose was 200-μg 89Zr-AMG 211 + 1,800-μg cold AMG 211. At 3 hours, the highest blood pool standardized uptake value (SUV)mean was 4.0, and tracer serum half-life was 3.3 hours. CD3-mediated uptake was clearly observed in CD3-rich lymphoid tissues including spleen and bone marrow (SUVmean 3.2 and 1.8, respectively), and the SUVmean decreased more slowly than in other healthy tissues. 89Zr-AMG 211 remained intact in plasma and was excreted predominantly via the kidneys in degraded forms. Of 43 visible tumor lesions, 37 were PET quantifiable, with a SUVmax of 4.0 [interquartile range (IQR) 2.7–4.4] at 3 hours using the optimal imaging dose. The tracer uptake differed between tumor lesions 5-fold within and 9-fold between patients. During AMG 211 treatment, tracer was present in the blood pool, whereas tumor lesions were not visualized, possibly reflecting target saturation.Conclusions:This first-in-human study shows high, specific 89Zr-AMG 211 accumulation in CD3-rich lymphoid tissues, as well as a clear, inter- and intraindividual heterogeneous tumor uptake.

Supplementary figure legend

Purpose: We evaluated biodistribution and tumor targeting of 89Zr-lumretuzumab before and during treatment with lumretuzumab, a human epidermal growth factor receptor 3 (HER3)–targeting monoclonal antibody.Experimental Design: Twenty patients with histologically confirmed HER3-expressing tumors received 89Zr-lumretuzumab and underwent positron emission tomography (PET). In part A, 89Zr-lumretuzumab was given with additional, escalating doses of unlabeled lumretuzumab, and scans were performed 2, 4, and 7 days after injection to determine optimal imaging conditions. In part B, patients were scanned following tracer injection before (baseline) and after a pharmacodynamic (PD)-active lumretuzumab dose for saturation analysis. HER3 expression was determined immunohistochemically in skin biopsies. Tracer uptake was calculated as standardized uptake value (SUV).Results: Optimal PET conditions were found to be 4 and 7 days after administration of 89Zr-lumretuzumab with 100-mg unlabeled lumretuzumab. At baseline using 100-mg unlabeled lumretuzumab, the tumor SUVmax was 3.4 (±1.9) at 4 days after injection. SUVmean values for normal blood, liver, lung, and brain tissues were 4.9, 6.4, 0.9 and 0.2, respectively. Saturation analysis (n = 7) showed that 4 days after lumretuzumab administration, tumor uptake decreased by 11.9% (±8.2), 10.0% (±16.5), and 24.6% (±20.9) at PD-active doses of 400, 800, and 1,600 mg, respectively, when compared with baseline. Membranous HER3 was completely downregulated in paired skin biopsies already at and above 400-mg lumretuzumab.Conclusions: PET imaging showed biodistribution and tumor-specific 89Zr-lumretuzumab uptake. Although, PD-active lumretuzumab doses decreased 89Zr-lumretuzumab uptake, there was no clear evidence of tumor saturation by PET imaging as the tumor SUV did not plateau with increasing doses. Clin Cancer Res; 23(20); 6128–37. ©2017 AACR.

Purpose: Mesothelin (MSLN) is frequently overexpressed in pancreatic and ovarian cancers, making it a potential drug target. We performed an 89Zr-PET imaging study with MMOT0530A, a MSLN antibody, in conjunction with a phase I study with the antibody–drug conjugate DMOT4039A, containing MMOT0530A bound to MMAE. The aim was to study antibody tumor uptake, whole-body distribution, and relation between uptake, response to treatment, and MSLN expression.Experimental Design: Before DMOT4039A treatment, patients received 37 MBq 89Zr-MMOT0530A followed by PET/CT imaging 2, 4, and 7 days postinjection. Tracer uptake was expressed as standardized uptake value (SUV). MSLN expression was determined with immunohistochemistry (IHC) on archival tumor tissue.Results: Eleven patients were included, 7 with pancreatic and 4 with ovarian cancer. IHC MSLN expression varied from absent to strong. Suitable tracer antibody dose was 10 mg MMOT0530A and optimal imaging time was 4 and 7 days postinjection. Tumor tracer uptake occurred in 37 lesions with mean SUVmax of 13.1 (±7.5) on PET 4 days postinjection, with 11.5 (±7.5) in (N = 17) pancreatic and 14.5 (±8.7) in (N = 20) ovarian cancer lesions. Within patients, a mean 2.4-fold (±1.10) difference in uptake between tumor lesions existed. Uptake in blood, liver, kidneys, spleen, and intestine reflected normal antibody distribution. Tracer tumor uptake was correlated to IHC. Best response to DMOT4039A was partial response in one patient.Conclusions: With 89Zr-MMOT0530A-PET, pancreatic and ovarian cancer lesions as well as antibody biodistribution could be visualized. This technique can potentially guide individualized antibody-based treatment. Clin Cancer Res; 22(7); 1642–52. ©2015 AACR.

Supplemental Figures and Tables

Purpose:CX-072, a PD-L1–targeting Probody therapeutic, is engineered to be activated by tumor proteases that remove a masking peptide. To study effects on biodistribution and pharmacokinetics, we performed 89Zr-CX-072 positron emission tomography (PET) imaging.Experimental Design:Patients received ∼1 mg, 37 MBq 89Zr-CX-072 plus 0, 4, or 9 mg unlabeled CX-072 and PET scans at days 2, 4, and 7. After that, treatment comprised 10 mg/kg CX-072 q2 weeks (n = 7) + 3 mg/kg ipilimumab q3w 4× (n = 1). Normal organ tracer uptake was expressed as standardized uptake value (SUV)mean and tumor uptake as SUVmax. PD-L1 expression was measured immunohistochemically in archival tumor tissue.Results:Three of the eight patients included received 10-mg protein dose resulting in a blood pool mean SUVmean ± SD of 4.27 ± 0.45 on day 4, indicating sufficient available tracer. Tumor uptake was highest at day 7, with a geometric mean SUVmax 5.89 (n = 113) and present in all patients. The median follow-up was 12 weeks (4–76+). One patient experienced stable disease and two patients a partial response. PD-L1 tumor expression was 90% in one patient and ≤1% in the other patients. Mean SUVmean ± SD day 4 at 10 mg in the spleen was 8.56 ± 1.04, bone marrow 2.21 ± 0.46, and liver 4.97 ± 0.97. Four patients out of seven showed uptake in normal lymph nodes and Waldeyer's ring. The tracer was intact in the serum or plasma.Conclusions:89Zr-CX-072 showed tumor uptake, even in lesions with ≤1% PD-L1 expression, and modest uptake in normal lymphoid organs, with no unexpected uptake in other healthy tissues.

Supplementary Figures 7-8 from VEGF-PET Imaging Is a Noninvasive Biomarker Showing Differential Changes in the Tumor during Sunitinib Treatment

In solid tumors, angiogenesis occurs in the setting of a defective vasculature and impaired lymphatic drainage that is associated with increased vascular permeability and enhanced tumor permeability. These universal aspects of the tumor microenvironment can have a marked influence on intratumoral drug delivery that may often be underappreciated. In this study, we investigated the effect of blood vessel normalization in tumors by the antiangiogenic drug bevacizumab on antibody uptake by tumors. In mouse xenograft models of human ovarian and esophageal cancer (SKOV-3 and OE19), we evaluated antibody uptake in tumors by positron emission tomographic imaging 24 and 144 hours after injection of 89Zr-trastuzumab (SKOV-3 and OE19), 89Zr-bevacizumab (SKOV-3), or 89Zr-IgG (SKOV-3) before or after treatment with bevacizumab. Intratumor distribution was assessed by fluorescence microscopy along with mean vessel density (MVD) and vessel normalization. Notably, bevacizumab treatment decreased tumor uptake and intratumoral accumulation compared with baseline in the tumor models relative to controls. Bevacizumab treatment also reduced MVD in tumors and increased vessel pericyte coverage. These findings are clinically important, suggesting caution in designing combinatorial trials with therapeutic antibodies due to a possible reduction in tumoral accumulation that may be caused by bevacizumab cotreatment. Cancer Res; 73(11); 3347–55. ©2013 AACR.

Supplementary Figure Legends 1-7 from VEGF-PET Imaging Is a Noninvasive Biomarker Showing Differential Changes in the Tumor during Sunitinib Treatment

Supplementary Figures 5-6 from VEGF-PET Imaging Is a Noninvasive Biomarker Showing Differential Changes in the Tumor during Sunitinib Treatment

PDF file - 53K, Biodistribution of 89Zr-Trastuzumab in the OE19 model and correlation of the in vivo PET tumor uptake with the ex vivo biodistriution tumor uptake of 89Zr-trastuzumab in the OE19 model.

PDF file - 58K

Supplementary Figures 1-2 from VEGF-PET Imaging Is a Noninvasive Biomarker Showing Differential Changes in the Tumor during Sunitinib Treatment

Supplementary Figures 3-4 from VEGF-PET Imaging Is a Noninvasive Biomarker Showing Differential Changes in the Tumor during Sunitinib Treatment

PURPOSE Testicular cancer (TC) treatment is clearly associated with cardiovascular morbidity and mortality. To enable development of preventive strategies for cardiovascular disease (CVD), we assessed cardiometabolic risk factors and quality of life (QoL) in TC survivors. METHODS Incidence of coronary artery disease, myocardial infarction, and heart failure after TC treatment was assessed in a multicenter cohort comprising 4,748 patients treated at the age of 12-50 years between 1976 and 2007. Patients who had developed CVD and a random sample from the cohort (subcohort) received a questionnaire on cardiometabolic risk factors and QoL. A subgroup of responders in the subcohort additionally underwent clinical evaluation of cardiovascular risk factors. RESULTS After a median follow-up of 16 years, 272 patients had developed CVD. Compared with orchidectomy only, cisplatin combination chemotherapy was associated with an increased CVD risk (hazard ratio [HR], 1.9; 95% CI, 1.1 to 3.1). Patients who were obese or a smoker at diagnosis (HR, 4.6; 95% CI, 2.0 to 10.0 and HR, 1.7; 95% CI, 1.1 to 2.4, respectively), developed Raynaud's phenomenon (HR, 1.9; 95% CI, 1.1 to 3.6) or dyslipidemia (HR, 2.8; 95% CI, 1.6 to 4.7) or had a positive family history for CVD (HR, 2.9; 95% CI, 1.7 to 4.9) had higher CVD risk. More TC survivors with CVD reported inferior QoL on physical domains than survivors who did not develop CVD. Of 304 TC survivors who underwent clinical evaluation for cardiovascular risk factors (median age at assessment: 51 years), 86% had dyslipidemia, 50% had hypertension, and 35% had metabolic syndrome, irrespective of treatment. CONCLUSION Cardiovascular events in TC survivors impair QoL. Many TC survivors have undetected cardiovascular risk factors. We advocate early lifestyle adjustments and lifelong follow-up with low-threshold treatment of cardiovascular risk factors, especially in obese and smoking patients treated with platinum-based chemotherapy.

PurposeAromatase inhibitors (AIs) are an important component of the adjuvant treatment of hormone receptor positive breast cancer (BC) but concerns regarding their cardiovascular safety remain. In this cross-sectional study nested in a breast cancer cohort, we investigated the association between AI exposure and early markers for cardiovascular disease in BC survivors.MethodsThe study population consisted of 569 women, who were 5-7 years (n=277) or 10-12 years (n=292) after BC diagnosis. All participants underwent carotid ultrasound, skin autofluorescence measurement and laboratory evaluation. To quantify AI exposure, we obtained the AI ratio by dividing the duration of AI use by the total duration of endocrine therapy (ET). Patients were classified according to their AI ratio into low (no ET or AI ratio 0.60). The association between AI ratio and carotid intima media thickness (cIMT), advanced glycation end products (AGEs) and the presence of dyslipidemia was assessed using linear and logistic regression.Results Median age at study visit was 55.5 years (range 45.2-63.8). Forty percent (n=231) of the study population had used AIs, of whom the majority sequentially with tamoxifen; median duration of AI use was 3.0 years. Mean cIMT and mean AGEs did not differ across AI exposure groups in univariable and multivariable analysis. The occurrence of dyslipidemia did not vary across AI exposure groups. Intermediate AI exposure was associated with more frequent occurrence of the combined endpoint (elevated cIMT, elevated AGEs and/or dyslipidemia). This association, however, was not present in the group with highest AI exposure.ConclusionAI exposure was not associated with cIMT, AGEs or the presence of dyslipidemia. Our results are reassuring for the cardiovascular safety of patients who receive treatment with AIs to reduce the risk of BC recurrence.Trial registration number (clinicaltrials.gov): NCT02485626, June 30, 2015

Immune checkpoint inhibitors (ICIs), by reinvigorating CD8+ T cell mediated immunity, have revolutionized cancer therapy. Yet, the systemic CD8+ T cell distribution, a potential biomarker of ICI response, remains poorly characterized. We assessed safety, imaging dose and timing, pharmacokinetics and immunogenicity of zirconium-89-labeled, CD8-specific, one-armed antibody positron emission tomography tracer 89ZED88082A in patients with solid tumors before and ~30 days after starting ICI therapy (NCT04029181). No tracer-related side effects occurred. Positron emission tomography imaging with 10 mg antibody revealed 89ZED88082A uptake in normal lymphoid tissues, and tumor lesions across the body varying within and between patients two days after tracer injection (n = 38, median patient maximum standard uptake value (SUVmax) 5.2, IQI 4.0–7.4). Higher SUVmax was associated with mismatch repair deficiency and longer overall survival. Uptake was higher in lesions with stromal/inflamed than desert immunophenotype. Tissue radioactivity was localized to areas with immunohistochemically confirmed CD8 expression. Re-imaging patients on treatment showed no change in average (geometric mean) tumor tracer uptake compared to baseline, but individual lesions showed diverse changes independent of tumor response. The imaging data suggest enormous heterogeneity in CD8+ T cell distribution and pharmacodynamics within and between patients. In conclusion, 89ZED88082A can characterize the complex dynamics of CD8+ T cells in the context of ICIs, and may inform immunotherapeutic treatments.

Purpose: CX-072, a PD-L1–targeting Probody therapeutic, is engineered to be activated by tumor proteases that remove a masking peptide. To study effects on biodistribution and pharmacokinetics, we performed 89Zr-CX-072 positron emission tomography (PET) imaging. Experimental Design: Patients received ∼1 mg, 37 MBq 89Zr-CX-072 plus 0, 4, or 9 mg unlabeled CX-072 and PET scans at days 2, 4, and 7. After that, treatment comprised 10 mg/kg CX-072 q2 weeks (n = 7) + 3 mg/kg ipilimumab q3w 4× (n = 1). Normal organ tracer uptake was expressed as standardized uptake value (SUV)mean and tumor uptake as SUVmax. PD-L1 expression was measured immunohistochemically in archival tumor tissue. Results: Three of the eight patients included received 10-mg protein dose resulting in a blood pool mean SUVmean ± SD of 4.27 ± 0.45 on day 4, indicating sufficient available tracer. Tumor uptake was highest at day 7, with a geometric mean SUVmax 5.89 (n = 113) and present in all patients. The median follow-up was 12 weeks (4–76+). One patient experienced stable disease and two patients a partial response. PD-L1 tumor expression was 90% in one patient and ≤1% in the other patients. Mean SUVmean ± SD day 4 at 10 mg in the spleen was 8.56 ± 1.04, bone marrow 2.21 ± 0.46, and liver 4.97 ± 0.97. Four patients out of seven showed uptake in normal lymph nodes and Waldeyer's ring. The tracer was intact in the serum or plasma. Conclusions: 89Zr-CX-072 showed tumor uptake, even in lesions with ≤1% PD-L1 expression, and modest uptake in normal lymphoid organs, with no unexpected uptake in other healthy tissues.

Background Adjuvant endocrine therapy (ET) in patients with breast cancer (BC) increases the risk of becoming less physically active. Physical inactivity is associated with a higher risk of treatment-related side effects and mortality. This study investigated whether supervised exercise increased the proportion of patients adhering to the national physical activity (PA) guideline during adjuvant ET in overweight or obese BC patients. Methods This multicentre single-arm clinical trial included patients with BC participating in a 12-week supervised exercise intervention. An accelerometer measured moderate to vigorous PA (MVPA) at baseline (T0), after 12 (T1) and 26 weeks (T2). The primary endpoint was change in the proportion of patients with weekly ≥150 min of MVPA at T1 compared to T0. Secondary endpoints were adherence to PA guideline at T2, metabolic syndrome (MetS), body composition, health-related quality of life (HRQoL) and BC-specific functioning and symptoms, self-reported PA, self-efficacy, exercise motivation and satisfaction with life. Results 141 patients with a median age of 61 years and a mean BMI of 31.3 participated. Adherence to the PA guideline increased from 38.3% at T0, to 40.4% at T1 (p = .112) and 44.7% at T2 (p = .003). MetS, body composition, HRQoL, BC-specific functioning and symptoms (i.e. fatigue, dyspnoea), self-reported PA, self-efficacy, exercise motivation and satisfaction with life improved significantly over time. Conclusions Supervised exercise increased the proportion of BC patients adhering to the PA guideline over time. Furthermore, MetS, body composition, HRQoL and symptoms improved. Our findings highlight the clinical relevance of supervised exercise during ET in overweight BC patients. Clinical trial information (NCT02424292)., Highlights • Overweight breast cancer patients on endocrine therapy can be motivated for supervised exercise. • Supervised exercise increases adherence to the physical activity guideline. • Metabolic syndrome and quality of life improved due to our intervention. • Participation in supervised exercise induces persistent clinical improvements.

PURPOSE The classification of the International Germ Cell Cancer Collaborative Group (IGCCCG) plays a pivotal role in the management of metastatic germ cell tumors but relies on data of patients treated between 1975 and 1990. MATERIALS AND METHODS Data on 9,728 men with metastatic nonseminomatous germ cell tumors treated with cisplatin- and etoposide-based first-line chemotherapy between 1990 and 2013 were collected from 30 institutions or collaborative groups in Europe, North America, and Australia. Clinical trial and registry data were included. Primary end points were progression-free survival (PFS) and overall survival (OS). The survival estimates were updated for the current era. Additionally, a novel prognostic model for PFS was developed in 3,542 patients with complete information on potentially relevant variables. The results were validated in an independent data set. RESULTS Compared with the original IGCCCG publication, 5-year PFS remained similar in patients with good prognosis with 89% (87%-91%) versus 90% (95% CI, 89 to 91), but the 5-year OS increased from 92% (90%-94%) to 96% (95%-96%). In patients with intermediate prognosis, PFS remained similar with 75% (71%-79%) versus 78% (76%-80%) and the OS increased from 80% (76%-84%) to 89% (88%-91%). In patients with poor prognosis, the PFS increased from 41% (95% CI, 35 to 47) to 54% (95% CI, 52 to 56) and the OS from 48% (95% CI, 42 to 54) to 67% (95% CI, 65 to 69). A more granular prognostic model was developed and independently validated. This model identified a new cutoff of lactate dehydrogenase at a 2.5 upper limit of normal and increasing age and presence of lung metastases as additional adverse prognostic factors. An online calculator is provided ( https://www.eortc.org/IGCCCG-Update ). CONCLUSION The IGCCCG Update model improves individual prognostication in metastatic nonseminomatous germ cell tumors. Increasing age and lung metastases add granularity to the original IGCCCG classification as adverse prognostic factors.

PURPOSE Patients with testicular germ cell tumor (TGCT) are at increased risk of developing a contralateral TGCT (CTGCT). Although some studies suggest that prior treatment with platinum-based chemotherapy affects CTGCT risk, a relationship between CTGCT risk and platinum dose has not previously been assessed. We analyzed the association between the number of platinum-based chemotherapy cycles and CTGCT risk. PATIENTS AND METHODS The risk of developing a metachronous CTGCT was evaluated in a nationwide cohort of 4,755 patients diagnosed with primary TGCT in the Netherlands between 1989 and 2007. Standardized incidence ratios were computed to compare CTGCT incidence with expected TGCT on the basis of TGCT incidence in the general population. The cumulative incidence of CTGCT was estimated in the presence of death as competing risk. The effect of treatment with platinum-based chemotherapy on CTGCT risk was assessed using multivariable Cox proportional hazards regression models. RESULTS CTGCT was diagnosed in 136 patients (standardized incidence ratio, 14.6; 95% CI, 12.2 to 17.2). The cumulative incidence increased up to 20 years after primary diagnosis, reaching 3.4% (95% CI, 2.8% to 4.0%) after 20 years of follow up. The risk of developing a CTGCT decreased with age (hazard ratio [HR], 0.93; 95% CI, 0.90 to 0.96), was lower after nonseminomatous germ cell tumor (HR, 0.58; 95% CI, 0.35 to 0.96) and decreased with every additional cycle of chemotherapy (HRper cycle, 0.74; 95% CI, 0.64 to 0.85). CONCLUSION Approximately one in every 30 survivors of TGCT will develop a CTGCT, with CTGCT incidence increasing up to 20 years after a primary TGCT. Treatment with platinum-based chemotherapy shows a dose-dependent inverse association with CTGCT risk.

Background The coronavirus disease 2019 (COVID-19) pandemic has become a public health emergency affecting frail populations, including patients with cancer. This poses the question of whether cancer treatments can be postponed or modified without compromising their efficacy, especially for highly curable cancers such as germ cell tumors (GCTs). Materials and Methods To depict the state-of-the-art management of GCTs during the COVID-19 pandemic, a survey including 26 questions was circulated by e-mail among the physicians belonging to three cooperative groups: (a) Italian Germ Cell Cancer Group; (b) European Reference Network–Rare Adult Solid Cancers, Domain G3 (rare male genitourinary cancers); and (c) Genitourinary Medical Oncologists of Canada. Percentages of agreement between Italian respondents (I) versus Canadian respondents (C), I versus European respondents (E), and E versus C were compared by using Fisher's exact tests for dichotomous answers and chi square test for trends for the questions with three or more options. Results Fifty-three GCT experts responded to the survey: 20 Italian, 6 in other European countries, and 27 from Canada. Telemedicine was broadly used; there was high consensus to interrupt chemotherapy in COVID-19–positive patients (I = 75%, C = 55%, and E = 83.3%) and for use of granulocyte colony-stimulating factor primary prophylaxis for neutropenia (I = 65%, C = 62.9%, and E = 50%). The main differences emerged regarding the management of stage I and stage IIA disease, likely because of cultural and geographical differences. Conclusion Our study highlights the common efforts of GCT experts in Europe and Canada to maintain high standards of treatment for patients with GCT with few changes in their management during the COVID-19 pandemic.

Treatment of stage II–IV nonseminomatous testicular germ cell tumors (NSTGCTs) consists of cisplatin-based combination chemotherapy and, when present, resection of residual retroperitoneal tumor mass (RRTM) by conventional laparotomy or laparoscopy. In case of a retroperitoneal recurrence, a second conventional or laparoscopic procedure may be challenging. A case of late relapse after prior conventional resection of a RRTM and tailor-made surgical management with a posterior retroperitoneoscopic resection (PRR) is reported. A posterior retroperitoneoscopic RRTM resection was performed in a 26-year-old male with a history of stage IIC NSTGCT, presenting with a late left-sided retroperitoneal relapse, 6 years after initial treatment. Postoperative course was uneventful and at 1-year follow-up the patient had no evidence of disease. Reoperative surgery by a minimal invasive retroperitoneoscopic approach should be considered as an alternative for patients with a recurrent retroperitoneal tumor mass of a NSTGCT.

SummaryBackground Metformin use is associated with reduced cancer risk in epidemiological studies and has preclinical anti-cancer activity in ovarian cancer models. The primary objective of this phase I study was to determine the recommended phase II dose (RP2D) of metformin in combination with carboplatin/paclitaxel in patients with ovarian cancer. Secondary objectives were to describe safety and pharmacokinetics. Methods In this single-center trial the RP2D of metformin in combination with carboplatin area under the concentration-time curve (AUC) 6 and paclitaxel 175 mg/m2 every 3 weeks (q3w) in patients with advanced epithelial ovarian cancer was determined using a 3 + 3 escalation rule at three fixed dose levels: 500 mg three times daily (tds), 850 mg tds and 1000 mg tds. Metformin was commenced on day 3 of cycle 1 and continued until 3 weeks after the last chemotherapy administration. The RP2D was defined as the dose level at which 0 of 3 or ≤ 1 of 6 evaluable subjects experienced a metformin-related dose-limiting toxicity (DLT). Safety was assessed according to CTCAE v4.0. Plasma and serum samples for pharmacokinetic (PK) analyses were collected during treatment cycles 1 and 2. Results Fifteen patients with epithelial ovarian cancer and an indication for neo-adjuvant (n = 5) or palliative (n = 10) treatment were included. No DLTs were observed. Three patients discontinued study treatment during cycle 1 for other reasons than DLT. Six patients were treated at the RP2D of metformin 1000 mg tds. The most frequent low-grade toxicities were anemia, hypomagnesemia and diarrhea. Grade 3 adverse events (AEs) occurred in ten patients, most common were leucopenia (n = 4), thrombocytopenia (n = 3) and increased GGT (n = 3). There were no grade 4 AEs. Metformin increased the platinum (Pt) AUC (Δ22%, p = 0.013) and decreased the Pt clearance (Δ-28%, p = 0.013). Metformin plasma levels were all within the therapeutic range for diabetic patients (0.1–4 mg/L). Conclusion The RP2D of metformin in combination with carboplatin and paclitaxel in advanced ovarian cancer is 1000 mg tds. This is higher than the RP2D reported for combination with targeted agents. A potential PK interaction of metformin with carboplatin was identified.

Aromatase inhibitors (AIs) are an important component of the adjuvant treatment of hormone receptor positive breast cancer (BC) but concerns regarding their cardiovascular safety remain. In this cross-sectional study nested in a breast cancer cohort, we investigated the association between AI exposure and early markers for cardiovascular disease in BC survivors.The study population consisted of 569 women, who were 5-7 years (n = 277) or 10-12 years (n = 292) after BC diagnosis. All participants underwent carotid ultrasound, skin autofluorescence measurement and laboratory evaluation. To quantify AI exposure, we obtained the AI ratio by dividing the duration of AI use by the total duration of endocrine therapy (ET). Patients were classified according to their AI ratio into low (no ET or AI ratio 0.40), intermediate (0.40 ≤ AI ratio ≤ 0.60) or high AI exposure (AI ratio 0.60). The association between AI ratio and carotid intima media thickness (cIMT), advanced glycation end products (AGEs) and the presence of dyslipidemia was assessed using linear and logistic regression.Median age at study visit was 55.5 years (range 45.2-63.8). Forty percent (n = 231) of the study population had used AIs, of whom the majority sequentially with tamoxifen; median duration of AI use was 3.0 years. Mean cIMT and mean AGEs did not differ across AI exposure groups in univariable and multivariable analysis. The occurrence of dyslipidemia did not vary across AI exposure groups. Intermediate AI exposure was associated with more frequent occurrence of the combined endpoint (elevated cIMT, elevated AGEs and/or dyslipidemia). This association, however, was not present in the group with highest AI exposure.AI exposure was not associated with cIMT, AGEs or the presence of dyslipidemia. These results do not prompt a change in current clinical practice, although further research is warranted to validate our findings over time and in different BC populations. Trial registration number (clinicaltrials.gov): NCT02485626, June 30, 2015.

Immune checkpoint inhibitors (ICIs), by reinvigorating CD8

Background Immune checkpoint inhibitors (ICIs) can induce durable responses in multiple different tumor types. Lymphocyte activation gene-3 (LAG-3) is one of the immune checkpoints for which therapeutic antibodies are being developed. PET imaging with radiolabeled ICIs can visualize whether the drug reaches the tumor, and might serve as a target expression readout. Therefore, we aimed to evaluate the safety of the radiolabeled LAG-3 antibody 89Zr-DFO-REGN3767, to determine the optimal tracer dose and imaging time point, and to get insight into 89Zr-DFO-REGN3767 whole body distribution. Methods Patients with advanced solid tumors received 37 MBq 89Zr-DFO-REGN3767 together with unlabeled antibody to achieve a 2, 5, 10, 20, or 40 mg total protein dose (NCT04706715). Patients underwent PET/CT scans on days 0, 2, 4, and 7 after injection, followed by a tumor biopsy. After that, they received treatment with cemiplimab alone or combined with platinum-containing chemotherapy. PET scans were analyzed by placing volumes of interest (VOIs) in tumor lesions and normal tissues. Tracer uptake was measured in the VOIs and expressed as the maximum standardized uptake value (SUVmax) for tumor lesions and as the mean standardized uptake value (SUVmean) for normal tissues. Results The 16 included patients experienced no 89Zr-DFO-REGN3767-related side effects. Tumor-to-blood ratios for all dose levels increased from days 0 to 7. Therefore, imaging 7 days after tracer injection was deemed optimal to achieve the highest contrast. The 40 mg tracer dose resulted in the most favorable blood kinetics. At this dose, tracer uptake in tumor lesions (n = 17) varied between patients (geometric mean SUVmax 6.2; SD 1.7). In normal tissues, uptake was seen in the spleen (x̄ 11.2; SD 1.6), and bone marrow (x̄ 2.2; SD 0.7), which increased from days 0 to 7. Tracer uptake was visually present in regions with inflammation. Conclusion 89Zr-DFO-REGN3767 PET imaging is feasible and studied patients did not experience tracer related side effects. Optimal imaging results were achieved with the 40 mg protein dose and imaging on day 7 89Zr-DFO-REGN3767 shows specific accumulation in LAG-3 rich tissues and tumor lesions. Citation Format: Pim P. van de Donk, Lotte M. Smit, Joyce van Sluis, Wim Timens, Adrienne H. Brouwers, Marjolijn N. Lub- de Hooge, Sjoerd G. Elias, Jourik A. Gietema, Claudia A. van Winkel, JuAn Wang, Jason Giurleo, Thomas Uldrick, Hung Kam Cheung, Hilde Jalving, Sjoukje F. Oosting, Daan G. Knapen, Derk-Jan A. de Groot, Elisabeth G. de Vries. LAG-3 PET imaging in patients with cancer before immune checkpoint inhibitor therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5611.

Higher levels of physical activity are associated with a lower risk of cardiovascular disease in the general population. Whether the same holds for women who underwent treatment for breast cancer is unclear.The aim of this study was to evaluate the association between physical activity in a typical week in the past 12 months and cardiac dysfunction in breast cancer survivors.We used data from a cohort of breast cancer survivors who were treated at ages 40 to 50 years (N = 559). The association between physical activity and global longitudinal strain (GLS) and left ventricular ejection fraction (LVEF) was evaluated using both linear and modified Poisson regression analyses adjusted for relevant confounders.In total, 559 breast cancer survivors were included, with median age of 55.5 years and a median time since treatment of 10.2 years. GLS was less favorable in inactive survivors (-17.1%) than in moderately inactive (-18.4%), moderately active (-18.2%), and active survivors (-18.5%), with an adjusted significant difference for active versus inactive survivors (β = -1.31; 95% CI: -2.55 to -0.06)). Moderately active (n = 57/130) and active survivors (n = 87/124) had significantly lower risks of abnormal GLS (defined as-18%) compared with inactive survivors (n = 17/26) (RR: 0.65 [95% CI: 0.45-0.94] and RR: 0.61 [95% CI: 0.43-0.87], respectively). LVEF, in normal ranges in all activity categories, was not associated with physical activity.In long-term breast cancer survivors, higher physical activity levels were associated with improved GLS but not LVEF, with the relatively largest benefit for doing any activity versus none. This finding suggests that increasing physical activity may contribute to cardiovascular health benefits, especially in inactive survivors.

Background: Impaired cognition can be a late effect after treatment in long-term testicular cancer (TC) survivors, negatively affecting their daily life. However, little data is available beyond 20 years post-treatment. We assessed cognitive impairment in very long-term TC survivors after CT or RT and compared the results with stage I TC survivors and controls. Methods: In this cross-sectional multicenter cohort study, we enrolled TC survivors (treated with orchiectomy followed by CT or RT or orchiectomy only)—with a follow-up duration ≥ 20 years—and age-matched healthy controls. Cognitive testing included the Auditory Verbal Learning Test, Letter Fluency Test, Category Fluency Test, and Trail Making Test. We used fasting blood samples to assess the presence of hypogonadism and measured cardiovascular aging parameters, including carotid pulse wave velocity (c-PWV) and advanced glycation end products (AGEs). Results: We included 184 TC survivors (66 CT patients, 53 RT patients, and 65 orchiectomy-only patients) and 70 healthy controls. The median follow-up was 26 years (range: 20–42). TC survivors had a lower combined score of the cognitive tests (mean cumulative Z-score −0.85, 95% CI −1.39 to −0.33) compared to controls (mean 0.67, 95% CI −0.21 to 1.57, p &lt, 0.01). In univariate analysis, the presence of hypogonadism (β −1.50, p &lt, 0.01), high c-PWV (β −0.35, p = 0.09), and high AGEs (β −1.27, p = 0.02) were associated with lower cognitive scores, while only AGEs (β −1.17, p = 0.03) remained a significant predictor in multivariate analysis (Model R2 0.31, p &lt, 0.01). Conclusions: Long-term TC survivors performed worse on cognitive tests compared to controls. Physicians and patients should be informed about timely cardiovascular risk management and testosterone supplementation therapy during follow-up to reduce the risk of cognitive impairment. Trial Registration: NCT02572934.

Background: Chemo- and radiotherapy for breast cancer (BC) can lead to cardiotoxicity even years after the initial treatment. The pathophysiology behind these late cardiac effects is poorly understood. Therefore, we studied a large panel of biomarkers from different pathophysiological domains in long-term BC survivors, and compared these to matched controls. Methods and results: In total 91 biomarkers were measured in 688 subjects: 342 BC survivors stratified either to treatment with chemotherapy ± radiotherapy (n = 170) or radiotherapy alone (n = 172) and matched controls. Mean age was 59 ± 9 years and 65 ± 8 years for women treated with chemotherapy ± radiotherapy and radiotherapy alone, respectively, with a mean time since treatment of 11 ± 5.5 years. No biomarkers were differentially expressed in survivors treated with radiotherapy alone vs. controls (P for all >0.1). In sharp contrast, a total of 19 biomarkers were elevated, relative to controls, in BC survivors treated with chemotherapy ± radiotherapy after correction for multiple comparisons (P