Jana Obertova

PARP expression in germ cell tumours

Background Poly(ADP-ribose)polymerase (PARP) inhibitors represent a new class of promising drugs in anticancer therapy. Aims To evaluate PARP expression in testicular germ cell tumours (GCTs) and to correlate expression patterns with clinicopathological variables. Methods In this translational study, tumour specimens from 124 patients with GCTs (114 patients with testicular primary tumours and 10 with extragonadal GCTs) were identified. PARP expression was detected by immunohistochemistry using monoclonal antibodies, scored by the multiplicative quickscore (QS) method and compared to PARP expression in normal testicular tissue. Results We observed higher expression of PARP in testicular tumours compared to normal testicular tissue (mean QS=10.04 vs 3.31, p<0.0000001). Mean QS±SD for each histological subtype was as follows: intratubular germ cell neoplasia unclassified (IGCNU)=18.00±0.00, embryonal carcinoma=9.62±5.64, seminoma=9.74±6.51, yolk sac tumour=7.8±7.20, teratoma=5.87±5.34, and choriocarcinoma=4.50±8.33. The PARP overexpression (QS>9) was most often detected in IGCNU (100% of specimen with PARP overexpression), seminona (52.6%), embryonal carcinoma (47.0%), yolk sac tumour (33.3%), teratoma (26.7%) and choriocarcinoma (25.0%), compared to 1.9% of normal testicular tissue specimens. There was no association between PARP expression and clinical variables. Conclusions In this pilot study, we showed for the first time, that PARP is overexpressed in testicular germ cell tumours compared to normal testis.

Phase II study of everolimus in refractory testicular germ cell tumors

BACKGROUND Testicular germ cell tumors (TGCTs) represent a highly curable disease; however, a small proportion of patients develop disease recurrence. Loss of the tumor-suppressor gene phosphatase and tensin homolog marks the transition from intratubular germ cell neoplasia to invasive GCT and is correlated with disease progression. Inactivation of phosphatase and tensin homolog is associated with deregulation of the PI3K/Akt pathway and increased mammalian target of rapamycin signaling. This study aimed to determine the efficacy and toxicity of a mammalian target of rapamycin inhibitor, everolimus, in patients with refractory TGCTs. METHODS From December 2011 to February 2015, 15 patients with refractory GCTs were enrolled in the phase II study. All patients were pretreated with at least 2 cisplatin-based therapies; 4 tumors (26.7%) were absolutely refractory to cisplatin and 9 patients (60.0%) had visceral nonpulmonary metastases. Everolimus was administered at a dose of 10mg daily until progression or unacceptable toxicity. The primary end point was the objective response rate, according to Response Evaluation Criteria in Solid Tumors. RESULTS No objective response was observed, but 6 patients (40.0%) achieved 12-week progression-free survival. During a median follow-up period of 3.6 months (range: 1-35.1mo), all patients experienced disease progression and 11 patients (80.0%) died. Median progression-free survival was 1.7 months (95% CI: 1.1-4.0mo) and median overall survival was 3.6 months (95% CI: 2.0-11.0mo). CONCLUSIONS This study failed to achieve its primary end point and our data suggest limited efficacy of everolimus against unselected heavily pretreated refractory TGCTs. CONDENSED ABSTRACT Everolimus showed limited efficacy in unselected heavily pretreated refractory TGCTs. Prolonged disease stabilization could be achieved in selected patients.

Sunitinib in Patients with Cisplatin-Refractory Germ Cell Tumors

markers were measured every 6 weeks, and computed tomography (CT) scans of the chest, abdomen, and pelvis were evaluated every 12 weeks. CT or magnetic resonance imaging studies of the brain and bone scans were carried out where clinically indicated. 1 patient with teratocarcinoma achieved partial remission in the lungs and retroperitoneal lymph nodes as well as a decrease in alpha-fetoprotein (AFP) after 2 treatment cycles, but progressed after 4 cycles. Another patient with a teratoma component in the primary tumor and high levels of beta-human chorionic gonadotropin (b-hCG) at relapse, achieved partial remission in the lungs and lymph nodes after 2 treatment cycles; however rapid elevation of b-hCG was seen after 4 cycles. In the study reported by Oechsle et al. [1], 2 patients with non-seminoma achieved partial remission, however it is not clear if a teratoma component was present in these germ cell tumors. Several translational studies suggested that vascular endothelial growth factor (VEGF) might play an important role in the growth and progression of germ cell tumors [3, 4]. Substantially higher VEGF expression has been found in patients with germ cell tumors compared with normal testis tissue. VEGF overexpression was most often seen in teratoma components of such tumors [3]. Teratoma is the most frequent histological element in late-relapsing germ cell tumors. Recently, Ramasubbaiah et al. [5] observed that 5 of 8 patients with late relapse responded to a combination of oxaliplatin and bevacizumab. However the histology of the responding patients was not reported. All patients that responded had rising serum hCG and/or AFP levels. Radiographic responses were observed at the time of declining markers, and thus it was impossible to know if any teratoma component was part of the response (personal communication with L.H. Einhorn). Previously, we observed the efficacy of bevacizumab in the treatment of growing teratoma syndrome [6]. It was reported previously that monotherapy with sunitinib has limited efficacy in patients with heavily pretreated, unselected, refractory germ cell tumors [1, 2]. However it is of great interest to explore the molecular characteristics of responding patients as it may allow identification of a molecular profile suitable for sunitinib therapy or treatment with other targeted agents [1]. At the National Cancer Institute of Slovakia, we performed a 1-arm, single-stage, phase II study aimed at evaluating the efficacy and toxicity of sunitinib in patients with refractory germ cell tumors. The study was multicentric, with participation of 3 study centers in Slovakia. The Ethical Committee of the National Cancer Institute in Bratislava approved the study which was registered in the European Clinical Trials Database, EudraCT number 2007-004981-42. Those patients with radiological and/or serological proof of relapsed metastatic germ cell tumors, who were not eligible for curative chemotherapy or surgery and had failed at least 2 platinum-based regimens or 1 platinum regimen in the case of platinum-refractory disease or primary mediastinal nonseminomatous germ cell tumor, were eligible. All other standard inclusion and exclusion criteria for the study of salvage treatment in refractory germ cell tumor patients were applied. We planned to study 28 patients. However due to poor accrual, only 10 patients were included in the trial (July 2008 to December 2010). All patients gave informed consent. The median age was 32 years (range 19–55 years). All patients were platinum-refractory, with the exception of one. Patients were pretreated with a median of 3 cisplatin-containing therapies (range 3–6). Sunitinib was administered at a dose of 50 mg daily for 4 weeks, followed by 2 weeks off therapy. Sunitinib had limited efficacy in this treatment population with a median progression-free survival and overall survival of 10.8 and 12.9 weeks, respectively. RECIST 1.0 criteria and/ or tumor markers were used to evaluate the response. Tumor

Plasma Cytokines Correlated With Disease Characteristics, Progression-Free Survival, and Overall Survival in Testicular Germ-Cell Tumor Patients

Micro‐Abstract We performed a translational study and found a correlation among overall survival, progression‐free survival, and circulating cytokines in metastatic testicular germ‐cell tumor patients. Association between baseline clinicopathologic features and plasma cytokines was also assessed. Plasma cytokines could be a potential biomarker for identification of high‐risk patients. Background: Cytokines are the communicators of immune system and are involved in all immune responses. The aim of this study was to assess the correlation among plasma cytokines, patient and tumor characteristics, and clinical outcome in chemonaive testicular germ‐cell tumor (TGCT) patients. Patients and Methods: This study included 92 metastatic chemotherapy‐naive TGCT patients treated with platinum‐based chemotherapy from July 2010 to March 2014. Plasma was isolated before first administration of chemotherapy, and the concentration of 51 plasma cytokines were analyzed using multiplex bead arrays. Results: At a median follow‐up of 33.2 months (range, 0.1‐54.8 months), 10.9% of patients experienced disease progression, and 7.6% died. Several cytokines were associated with different baseline clinicopathologic features. Elevated plasma levels of interferon (IFN)‐&agr;2, interleukin (IL)‐2R&agr;, IL‐16, hepatocyte growth factor (HGF), and monocyte chemotactic protein (MCP)‐3 were significantly associated with worse progression‐free survival and overall survival (OS). Moreover, elevated levels of stem‐cell growth factor (SCGF)‐&bgr; were also associated with worse OS. Patients with elevated levels of all 6 cytokines experienced significantly worse outcomes compared to patients who had fewer than 6 cytokines elevated (hazard ratio = 12.06; 95% confidence interval, 7.39‐19.49; P = .002 for progression‐free survival, and hazard ratio = 39.65; 95% confidence interval, 25.03‐62.18; P < .00001 for OS, respectively). Results were independent of International Germ Cell Cancer Collaborative Group criteria. Conclusion: We found a correlation among progression free‐survival, OS, and circulating cytokines in TGCT. This suggests the existence an association between plasma cytokines and baseline clinicopathologic features in TGCT. Plasma cytokines could be used for identification of high‐risk patients who are candidates for new therapeutic approaches.

Paclitaxel, ifosfamide, and cisplatin (TIP) salvage therapy for patients (pts) with relapsed testicular germ cell tumors (GCT)

4632 Background: Using TIP with higher dose of paclitaxel (250mg/m2 ) Donadio et al (ASCO, 2003) achieved the optimal response in 74% of prognostic favorable GCT and 92% of them are relapse-free more than 2 years. We present our results in similar group of patients achieved by TIP with standard dose of paclitaxel. METHODS 17 pts received the TIP for 1. relaps after favorable response (complete response or a partial response with normal serum tumor markers ) to previous cisplatin containing therapy and in 14 pts the favorable response lasted > 6 months. 4 pts relapsed later than 2 years (late relapse). Four cycles of TIP were given 21 days apart. Paclitaxel 175mg/m2 was administered by 3-hour infusion on day 1, followed by ifosfamide 1.2 g/m2 and cisplatin 20 mg/m2on days 1 through 5. The administration of hemopoetic growth factors was not scheduled. Surgical resection of all radiographic residua was considered. RESULTS The favorable response was achieved in 10 (59%) pts and 3 of them relapsed. Two years estimated relapse free survival is 70%. Twelve pts (70%) remain alive at median f/u 39 months. 3 of 4 pts with late relapse achieved favorable response and two of them are being maintained from 11+ and 39+ months. Myelosuppression was the major toxicity, Gr3-4 leukopenia was experienced by 11% of courses. Nobody had Gr3-4 non-hematological toxicity. CONCLUSIONS TIP with standard dose of paclitaxel is effective and safe regimen for patients with prognostic favorable relapse of GCT. The number of durable response was lower than was achieved by higher dose of paclitaxel. No significant financial relationships to disclose.

The prognostic value of DNA damage level in peripheral blood lymphocytes of chemotherapy-naïve patients with germ cell cancer

Germ cell tumors (GCTs) are extraordinarily sensitive to cisplatin (CDDP)-based chemotherapy. DNA damage represents one of the most important factors contributing to toxic effects of CDDP-based chemotherapy. This study was aimed to evaluate the prognostic value of DNA damage level in peripheral blood lymphocytes (PBLs) from chemo-naïve GCT patients. PBLs isolated from 59 chemotherapy-naïve GCT patients were included into this prospective study. DNA damage levels in PBLs were evaluated by the Comet assay and scored as percentage tail DNA by the Metafer-MetaCyte analyzing software. The mean ± SEM (standard error of the mean) of endogenous DNA damage level was 5.25 ± 0.64. Patients with DNA damage levels lower than mean had significantly better progression free survival (hazard ratio [HR] = 0.19, 95% CI (0.04 – 0.96), P = 0.01) and overall survival (HR = 0.00, 95% CI (0.00 – 0.0), P < 0.001) compared to patients with DNA damage levels higher than mean. Moreover, there was significant correlation between the DNA damage level and presence of mediastinal lymph nodes metastases, IGCCCG (International Germ Cell Cancer Collaborative Group) risk group, and serum tumor markers level. These data suggest that DNA damage levels in PBLs of GCT patients may serve as an important prognostic marker early identifying patients with poor outcome.

Prognostic value of intratumoral carbonic anhydrase IX expression in testicular germ cell tumors

Testicular germ cell tumors (TGCTs) represent a highly curable malignancy, however a small proportion of patients fails to be cured with cisplatin-based chemotherapy. Carbonic anhydrase IX (CA IX) is upregulated by hypoxia in several cancer types and correlates with a poor prognosis. The present translational study evaluated expression and prognostic value of CA IX in TGCTs. Surgical specimens from 228 patients with TGCTs were processed by the tissue microarray method and subjected to immunohistochemistry with the M75 monoclonal antibody. CA IX expression was evaluated in tumors vs. adjacent normal testicular tissues and correlated with clinicopathological characteristics and clinical outcome. CA IX expression was detected in 62 (30.2%) of TGCTs compared to 0 (0%) of normal tissue adjacent to testicular tumor (P<0.001). The highest frequency of the CA IX expression was detected in teratoma (39.0%), followed by seminoma (22.7%), yolk sac tumor (22.2%), embryonal carcinoma (11.9%) and choriocarcinoma (7.7%). None of germ cell neoplasias in situ (GCNIS) exhibited CA IX expression. Patients without the CA IX tumor expression showed significantly better progression-free survival, but not overall survival, compared to patients with the CA IX expression [hazard ratio (HR), 0.57; 95% CI, 0.32–1.02; P=0.037 and HR, 0.58; 95% CI, 0.29–1.16; P=0.088, respectively]. There was no significant correlation between the CA IX expression and clinicopathological variables. The intratumoral CA IX expression can serve as a prognostic marker in the TGCT patients. These results suggest that activation of the hypoxia-induced pathways may be important in the treatment failure in TGCTs patients.

Prognostic value of serum carbonic anhydrase IX in testicular germ cell tumor patients

Despite the fact that testicular germ cell tumors (TGCTs) are one of the most chemosensitive solid tumors, a small proportion of patients fail to be cured following cisplatin-based first line chemotherapy. Upregulation of carbonic anhydrase IX (CA IX) in various solid tumors is associated with poor outcome. The current prospective study investigated the prognostic value of serum CA IX level in TGCTs. In total, 83 patients (16 non-metastatic following orchiectomy with no evidence of disease, 57 metastatic chemotherapy-naïve and 10 metastatic relapsed chemotherapy-pretreated) starting adjuvant and/or new line of chemotherapy and 35 healthy controls were enrolled in the study. Serum CA IX values were determined using an enzyme-linked immunosorbent assay, and intratumoral CA IX was analyzed by immunohistochemistry. Metastatic chemotherapy-naïve patients had significantly higher mean CA IX serum levels than healthy controls (490.6 vs. 249.6 pg/ml, P=0.005), while there was no difference in serum CA IX levels in non-metastatic or relapsed TGCT patients compared with healthy controls. There was no significant difference in the mean serum CA IX levels between different groups of patients and between the first and second cycle of chemotherapy, nor association with patients/tumor characteristics. Serum CA IX was not prognostic for progression-free survival [hazard ratio (HR)=0.81, P=0.730] or overall survival (HR=0.64, P=0.480). However, there was a significant association between intratumoral CA IX expression and serum CA IX concentration (rho=0.51, P=0.040). These results suggest that serum CA IX level correlates with tumor CA IX expression in TGCT patients, but fails to exhibit either a prognostic value or an association with patients/tumor characteristics.

[Is the same tyrosine kinase inhibitor still effective after development of brain metastases? A Case report].

There is a new era of treatment options since introduction of new biological targeted therapies (tyrosine kinase inhibitors (TKIs) and mammalian target of rapamycin (mTOR) inhibitors) in renal cell cancer. However, in patients who developed brain metastases, there is still treatment dilemma about an optimal therapeutic scenario, particularly in the subgroup of patients with-out disease progression outside the central nervous system. The objective of this case report is to present that it is possible to continue the same targeted therapy after development of brain metastasis after application of local whole brain irradiation with meaningful overall survival.

Antioxidant stimulation as the adaptation mechanism in patients with metastatic urothelial cancers (MUC) treated with gemcitabine and cisplatin (GC).

281 Background: The role of oxidative stress inpatients with MUC treated with GC is unknown. The objective of this ongoing study is to determinate plasma antioxidants, lipid peroxidation, and lipids in pts. with MUC before and after GC treatment, and to compare them with control. METHODS From May 2010 - January 2012, 42 MUC patients were enrolled into this study. The median of age was 65 years (range 44-81), median of metastasis sites was 2 (range 1-7). All patients were treated with gemcitabine 1000 mg/m2 i.v. days 1 and 8 with cisplatin 75 mg/m2 i.v. day 1, the new cycle of treatment started on day 22. Plasma samples were collected before and after 3 cycles of chemotherapy with GC. The control consisted of 22 subjects without cancer with age median 61 years (range 39-66). RESULTS In MUC patients, plasma γ-tocopherol before treatment were significantly lower (1.77±0.70 vs 2.13±0.70 μmol/L, P<0.04) and plasma TBARS higher vs control (5.76±1.32 vs 4.51±0.56 μmol/L, P<0.001). When compared baseline plasma levels to values after 3 cycles of GC, significant changes were noticed: γ-tocopherol (1.77±0.70 vs 2.29±1.14 μmol/L, P<0.04), α-tocopherol (24.23±5.14 vs 29.25±6.24 μmol/L, P<0.003), CoQ10-TOTAL (0.43±0.15 vs 0.56±0.16 μmol/L, P=0.003). TBARS concentrations were significantly decreased (5.76±1.32 vs 4.85±1.08 μmol/L, P=0.01). The concentrations of plasma lipids after GC were increased: Cholesterol (4.61±0.99 vs 5.47±1.21 mmol/L, P<0.004), TAG (1.30±0.65 vs 1.74±0.81 mmol/L, P<0.02), LDL cholesterol (2.94±0.73 vs 3.46±0.86 mmol/L, P<0.02), VLDL cholesterol (0.59±0.30 vs 0.81±0.37 mmol/L, P<0.02), and HDL cholesterol (1.08±0.39 vs 1.24±0.43 mmol/L, NS). CONCLUSIONS GC in patients with MUC stimulated antioxidant defence system, increased plasma lipids, and decreased lipid peroxidation. The antioxidant stimulation might be one of the adaptation mechanisms that protect organism and might prevent or reduce chemotherapy side-effects. Acknowledgement: Study is supported by VEGA 1/0614/12 and Ministry of Health, Slovakia, grant 2007/29-UK-06.