Frank Mayer

Maintaining success, reducing treatment burden, focusing on survivorship: highlights from the third European consensus conference on diagnosis and treatment of germ-cell cancer

In November 2011, the Third European Consensus Conference on Diagnosis and Treatment of Germ-Cell Cancer (GCC) was held in Berlin, Germany. This third conference followed similar meetings in 2003 (Essen, Germany) and 2006 (Amsterdam, The Netherlands) [Schmoll H-J, Souchon R, Krege S et al. European consensus on diagnosis and treatment of germ-cell cancer: a report of the European Germ-Cell Cancer Consensus Group (EGCCCG). Ann Oncol 2004; 15: 1377–1399; Krege S, Beyer J, Souchon R et al. European consensus conference on diagnosis and treatment of germ-cell cancer: a report of the second meeting of the European Germ-Cell Cancer Consensus group (EGCCCG): part I. Eur Urol 2008; 53: 478–496; Krege S, Beyer J, Souchon R et al. European consensus conference on diagnosis and treatment of germ-cell cancer: a report of the second meeting of the European Germ-Cell Cancer Consensus group (EGCCCG): part II. Eur Urol 2008; 53: 497–513]. A panel of 56 of 60 invited GCC experts from all across Europe discussed all aspects on diagnosis and treatment of GCC, with a particular focus on acute and late toxic effects as well as on survivorship issues. The panel consisted of oncologists, urologic surgeons, radiooncologists, pathologists and basic scientists, who are all actively involved in care of GCC patients. Panelists were chosen based on the publication activity in recent years. Before the meeting, panelists were asked to review the literature published since 2006 in 20 major areas concerning all aspects of diagnosis, treatment and follow-up of GCC patients, and to prepare an updated version of the previous recommendations to be discussed at the conference. In addition, ∼50 E-vote questions were drafted and presented at the conference to address the most controversial areas for a poll of expert opinions. Here, we present the main recommendations and controversies of this meeting. The votes of the panelists are added as online supplements.

Microsatellite Instability, Mismatch Repair Deficiency, and BRAF Mutation in Treatment-Resistant Germ Cell Tumors

PURPOSE Mismatch repair (MMR) deficiency and microsatellite instability (MSI) are associated with cisplatin resistance in human germ cell tumors (GCTs). BRAF mutation (V600E) is found in MSI colorectal cancers. The role of RAS/RAF pathway mutations in GCT treatment response is unknown. PATIENTS AND METHODS Two patient cohorts were investigated: 100 control GCTs (50 seminomas and 50 nonseminomas) and 35 cisplatin-based chemotherapy-resistant GCTs. MMR proteins were analyzed by immunohistochemistry, and eight microsatellite loci were examined for MSI. Tumors were assessed for specific BRAF and KRAS mutations. RESULTS Resistant tumors showed a higher incidence of MSI than controls: 26% versus 0% in two or more loci (P < .0001). All resistant tumors were wild-type KRAS, and two controls (2%) contained a KRAS mutation. There was a significantly higher incidence of BRAF V600E mutation in resistant tumors compared with controls: 26% versus 1% (P < .0001). BRAF mutations were highly correlated with MSI (P = .006), and MSI and mutated BRAF were correlated with weak or absent staining for hMLH1 (P = .017 and P = .008). Low or absent staining of hMLH1 was correlated with promoter hypermethylation (P < .001). Tumors lacking expression of hMLH1 or MSH6 were significantly more frequent in resistant GCTs than in controls (P = .001 and 0.0036, respectively). Within the subgroup of resistant tumors, patients with MSI showed a trend to longer progression-free survival (P = .068). CONCLUSION We report for the first time a correlation between a gene mutation--BRAF V600E--and cisplatin resistance in nonseminomatous GCTs. Furthermore, a correlation between MMR deficiency, MSI, and treatment failure is confirmed.

Role of P53 and MDM2 in treatment response of human germ cell tumors.

PURPOSE Testicular germ cell tumors (TGCTs) of adolescents and adults are very sensitive to systemic treatment. The exquisite chemosensitivity of these cancers has been attributed to a high level of wild-type P53. MATERIALS AND METHODS To clarify the role of P53 in treatment sensitivity and resistance of TGCTs, we performed immunohistochemistry and Western blotting analysis on a series of 39 fresh-frozen primary TGCTs before therapy (unselected series). In a series of formalin-fixed paraffin-embedded TGCTs of patients with fully documented clinical course, including treatment-sensitive (n = 17) and -resistant (n = 18) tumors, P53 status was assessed by immunohistochemistry and mutation analysis. In addition, the involvement of MDM2, a P53 antagonist, was investigated by immunohistochemistry, reverse transcriptase polymerase chain reaction, and in situ hybridization. RESULTS Immunohistochemistry demonstrated absence of staining for P53 in 36%, 41%, and 17% of the unselected, responding, and nonresponding TGCTs, respectively. Of the positive TGCTs, most tumors, ie, 49%, 41%, and 33%, showed 1% to 10% positive nuclei. This overall low level of P53 was confirmed by Western blotting. Mutation analysis revealed only one silent P53 mutation in one of the responding patients. All embryonal carcinomas were homogeneously positive for MDM2, encoded by the full length mRNA, while a heterogeneous pattern was found for the other histologic components. Amplification of MDM2 was detected in one out of 12 embryonal carcinomas. CONCLUSION Although our results are in line with previous findings of the presence of wild-type P53 in TGCTs, they show that a high level of P53 does not relate directly to treatment sensitivity of these tumors, and inactivation of P53 is not a common event in the development of cisplatin resistance.

Efficacy of cytotoxic agents used in the treatment of testicular germ cell tumours under normoxic and hypoxic conditions in vitro.

Platinum-based chemotherapy is the main treatment element to achieve cure for patients with metastatic germ cell tumours. Drug resistance in testicular germ cell tumours (TGCTs) is rare and the reasons are not fully understood. While recent investigations have indicated decreased efficacy of chemotherapy in several tumour types under hypoxic conditions, this aspect has not been investigated in TGCTs so far. Furthermore, for cisplatin – the most active drug in this disease – controversial effects of hypoxia on cytotoxic efficacy have been reported. The relative efficacy of cytotoxic agents for the treatment of TGCT patients was studied in three different cell lines derived from human embryonal carcinomas (EC) in an in vitro hypoxia model. NT2, 2102 EP, and NCCIT were tested for their sensitivity towards cisplatin, etoposide, bleomycin, 4-OOH-ifosfamide, carboplatin, paclitaxel, gemcitabine, oxaliplatin, irinotecan, and mitomycin C under normoxic and hypoxic conditions using the MTT assay. Inhibitory concentrations IC50 of the tested agents under both conditions were compared. Selected results were confirmed by flow-cytometric assessment of the apoptotic index. In all cells, doubling times were prolonged in hypoxia (NT2<NCCIT<2102 EP). All drugs were less effective under hypoxic conditions, including mitomycin C (eg, 1.6-fold increase of IC50 in hypoxia compared to normoxia for NT2) and cisplatin (eg, NT2: two-fold increase). The relative effect of hypoxia on the IC50 depended mainly on the cell line, and to a lesser extent on the drug. The results indicate that the reduced cell proliferation in hypoxia might be an important factor, but not the only determinant of a reduced cytotoxicity. In view of the broad spectrum of drugs with different modes of action tested, the relative resistance cannot be mediated by substance-specific resistance mechanisms like hypoxia-induced upregulation of P-glycoprotein or increased DNA-repair capacity, since many unrelated drugs were affected to a comparable extent in their efficacy by hypoxia. This study also provides the rationale to test the hypothesis whether improving tumour oxygenation by raising haemoglobin concentrations, for example, with erythropoietin in patients with TGCTs receiving chemotherapy may improve the outcome.

Late toxicity following curative treatment of testicular cancer

Cisplatin appears to be the major cause for long-term toxicity in patients treated for testicular cancer. Long-term side effects consist mainly of nephrotoxicity, ototoxicity, and neurotoxicity as well as gonadal damage. Following standard-dose chemotherapy approximately 20% to 30% of patients will be affected by long-term side effects, although not all these side effects will cause an impaired quality of life. Several strategies have been or currently are being evaluated to reduce acute and long-term complications including the introduction of equally effective, but less toxic regimens, or the use of cytoprotective agents such as amifostine. Secondary acute myeloid leukemia and secondary myelodysplastic syndrome probably represent the worst possible long-term complications of cancer therapy in those patients who originally were cured of their primary testicular cancer. Therapy-related solid tumors are mainly associated with the use of radiation therapy and the risk for developing a therapy-related solid tumor is increased approximately two to three times compared to the general population. In contrast, therapy-related leukemias are predominantly associated with chemotherapy, particularly with the use of topoisomerase-II inhibitors and alkylating agents. In general, the cumulative incidence of therapy-related leukemia following treatment of germ cell cancer is low. It is approximately 0.5% and 2% at 5 years of median follow-up for patients receiving etoposide at cumulative doses< or = 2 g/m(2) and >2 g/m(2), respectively. The risk-benefit analysis in patients with testicular cancer clearly favors the use of current treatment regimens including high-dose chemotherapy. However, even the acceptably low number of therapy-related long-term complications should encourage the search for equally effective but less toxic therapies. This review will highlight important available data about therapy-related toxicity and particularly, therapy-related malignancies following cisplatin-etoposide-based chemotherapy.

Germ cell lineage differentiation in non-seminomatous germ cell tumours

Human germ cell tumours (GCTs) have long fascinated investigators for a number of reasons. Being pluripotential tumours, they can differentiate into both extra‐embryonic and embryonic (somatic) tissues. However, it has never been shown convincingly that, in humans, these tumours are truly totipotent and can also give rise to the germ lineage, the third major differentiation lineage occurring early during embryonic life. Using a number of newly available, distinct, immunohistochemical markers, such as OCT3/4, VASA and TSPY, the occurrence of germ cells was investigated in a number of germ cell tumours. Development of germ cells was identified in three independent non‐seminomas, including two pure yolk sac tumours and one mixed tumour composed of yolk sac tumour and immature teratoma. Our finding indicates a previously unknown totipotent potential of human GCTs and raises the question of whether, under certain culture conditions, primordial germ cells could be derived from human GCT cell lines. Copyright

Preclinical and clinical activity of sunitinib in patients with cisplatin-refractory or multiply relapsed germ cell tumors: a Canadian Urologic Oncology Group/German Testicular Cancer Study Group cooperative study

BACKGROUND The objective of the study was to investigate the activity of sunitinib in a cell line model and subsequently in patients with cisplatin-refractory or multiply relapsed germ cell tumors (GCT). METHODS The effect of sunitinib on cell proliferation in cisplatin-sensitive and cisplatin-refractory GCT cell lines was evaluated after 48-h sunitinib exposure by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, and IC(50) (concentration that causes 50% inhibition of growth) doses were determined. Sunitinib was subsequently administered at a dose of 50 mg/day for 4 weeks followed by a 2-week break to 33 patients using a Simon two-stage design. RESULTS Sunitinib demonstrated comparable dose-dependent growth inhibition in cisplatin-sensitive and cisplatin-resistant cell lines, with IC(50) between 3.0 and 3.8 μM. Patient characteristics were as follows: median of 2 (1-6) cisplatin-containing regimens; high-dose chemotherapy 67%; late relapse 33%; and cisplatin refractory or absolute cisplatin refractory 54%. Toxic effects included fatigue (39%), anorexia (21%), diarrhea (27%), mucositis (45%), nausea (33%), hand-foot syndrome (12%), dyspepsia (27%), and skin rash (18%). No unexpected side-effects were observed. Thirty -two of 33 patients were assessable for response. Three confirmed partial responses (PRs) and one unconfirmed PR were seen for a total response rate of 13%. Median progression-free survival (PFS) was 2 months, with a 6-month PFS rate of 11%. CONCLUSIONS Sunitinib shows in vitro activity in cisplatin-resistant GCT cell lines. Modest clinical activity in heavily pretreated GCT patients was observed.

Differential changes in platelet VEGF, Tsp, CXCL12, and CXCL4 in patients with metastatic cancer

Data from animal studies indicate that platelets play a key role in tumor dissemination and metastasis. We therefore hypothesized that metastastic cancer patients may display a specific platelet phenotype. Percentage of activated, p-selectin positive platelets as well as platelet contents (i.e., plasma and platelet count-corrected serum levels of VEGF-A, CXCL12, CXCL4, and thrombospondin-1) were analyzed in 43 patients with newly diagnosed metastatic disease prior to treatment. Tumor patients had increased platelet counts and significantly elevated percentages of activated platelets. Moreover, the platelet content of VEGF-A in cancer patients was significantly increased compared to healthy controls, while thrombospondin-1, CXCL12 and CXCL4 were significantly decreased. Our data contain several unexpected results: firstly, CXCL12 was found in minute quantities in the serum as compared with murine studies. Secondly, CXCL4, which was found by mass spectrometry to be the single massively upregulated intraplatelet chemokine in mice after tumor xenotransplantation, was decreased in tumor patient platelets. While increased contents of VEGF-A have been attributed to platelet scavenger activity, the differential decrease of specific platelet contents may be due to differential secretion or altered megakaryopoiesis in metastatic cancer patients.

Aneuploidy of human testicular germ cell tumors is associated with amplification of centrosomes

Testicular germ cell tumors occur in three age groups. Seminomas and nonseminomas of adults, including mature teratomas, and the precursor carcinoma in situ (CIS) are aneuploid. This also holds true for yolk sac tumors of newborn and infants, while the mature teratomas of this age are diploid. In contrast, spermatocytic seminomas occurring in the elderly contain both diploid and polyploid cells. Aneuploidy has been associated with centrosome aberrations, sometimes related to overexpression of STK15. Aneuploidy of non-neoplastic germ cells has been demonstrated in the context of male infertility, a risk factor for the development of seminoma/nonseminoma. We investigated aneuploidy, centrosome aberrations and the role of STK15 in different types of testicular germ cell tumors as well as in normal and disturbed spermatogenesis. The aneuploid seminomas and nonseminomas tumors (including CIS) showed increased numbers of centrosomes, without STK15 amplification or overexpression. Four out of six infantile teratomas had normal centrosomes, the remaining two and an infantile yolk sac tumor showed a heterogeneous pattern of cells with normal or amplified centrosomes. Spermatocytic seminomas had two, four or eight centrosomes. Germ cells in seminiferous tubules with disturbed spermatogenesis shared both aneuploidy and centrosome abnormalities with seminomas/nonseminomas and showed a more intense STK15 staining than those with normal spermatogenesis and CIS. Therefore, aneuploidy of testicular germ cell tumors is associated with amplified centrosomes probably unrelated to STK15.

Long-Term Survival After Treatment with Gemcitabine and Oxaliplatin With and Without Paclitaxel Plus Secondary Surgery in Patients with Cisplatin-Refractory and/or Multiply Relapsed Germ Cell Tumors

BACKGROUND Chemotherapy including gemcitabine, oxaliplatin, and/or paclitaxel has shown efficacy in germ cell tumor patients after progression during cisplatin-based chemotherapy or relapse after high-dose chemotherapy including complete responses in 5-15%. OBJECTIVE Most studies have been published with a short follow-up. We present the long-term outcome of two previously reported trials. DESIGN, SETTING, AND PARTICIPANTS Two phase 2 trials have evaluated chemotherapy with gemcitabine plus oxaliplatin alone (GO) or plus paclitaxel (GOP) including a total of 76 patients (35 GO and 41 GOP). At first publication, 29 patients were still alive and 9 patients (12%) were free of disease after chemotherapy with or without surgery: GO, 3 of 35 (9%) and GOP, 6 of 41 (15%). MEASUREMENTS Survival and follow-up time were calculated using the Kaplan-Meier method from the beginning of study treatment until the date of death or the date of the last follow-up. RESULTS AND LIMITATIONS After a median follow-up of 19 mo (2-86 mo) for the 29 patients still alive, 11% of all patients (8 of 76) were free of disease for >2 yr: 1 of 35 patients (3%) after GO and 7 of 41 patients (17%) after GOP. Three patients with complete remission (CR), two after GO and one after GOP, relapsed. Two others treated with GOP were rendered disease free: One patient with partial remission and short follow-up underwent secondary surgery, and another patient, who had relapsed 2 mo after GOP, achieved a CR after salvage treatment. Overall survival time is ≥33 mo (range: ≥ 28-59 mo) in these eight patients. CONCLUSIONS Long-term survival can be achieved in about 10-15% of patients with cisplatin-refractory or multiply relapsed germ cell tumor with GO(P) chemotherapy. Aggressive secondary surgery following partial remission is a crucial part of this salvage treatment.