Katharina Prieske

Systemic treatment of vulvar cancer

Squamous cell carcinoma of the vulva is a rare disease, accounting for approximately 5% of cancers of the female genital tract. Standard therapy for early-stage vulvar cancer mainly comprises of surgery of the vulva and groins. In locally advanced or metastatic vulvar cancer, neoadjuvant or definitive chemoradiation is often considered as an alternative treatment option. Given its rarity, the level of evidence for different treatment modalities is poor and few clinical trials have been performed on this disease. Therefore indication criteria for systemic treatment in advanced stage vulvar cancer vary widely among countries and institutions. This review focuses on the different systemic treatment options for patients with locally advanced, recurrent or metastatic vulvar cancer, and highlights the need for an international multicenter approach to identify the most effective therapeutic options.

E-Cadherin fragments as potential mediators for peritoneal metastasis in advanced epithelial ovarian cancer

Background:Peritoneal dissemination and retroperitoneal lymph node involvement are main routes for tumour spread of epithelial ovarian cancer (EOC), possibly determined by the intercellular connecting protein E-Cadherin (E-Cad) and its fragments.Methods:Tumour tissue of 105 advanced EOC patients was evaluated for protein expression of E-Cad, β-Catenin and Calpain by western blotting and immunohistochemistry. Expression patterns were compared between tumours with solely intraperitoneal (pT3c, pN0; n=41) and tumours with retroperitoneal metastases (pT1a-3c, pN1; n=64). Lysates of the EOC cell line SKOV3 and tumour tissue from the intraperitoneal group were tested for E-Cad expression following Calpain treatment.Results:E-Cad full-length (E-Cad-FL, 120 kDa) and two major fragments at 85 kDa (E-Cad-85) and 23 kDa (E-Cad-23) were detected by western blotting. E-Cad-85 expression was significantly higher in tumours with solely intraperitoneal metastases and correlated strongly with E-Cad-23 and the protease Calpain. Calpain-mediated cleavage was identified as a potential mechanism to generate E-Cad-85 from E-Cad-FL by treating lysates from SKOV3 cells and tumour tissue with this enzyme. Increased cytoplasmic localisation of β-Catenin in tumours with high E-Cad-85 expression corroborates that E-Cad-85 loses the binding site for β-Catenin after fragmentation, enabling tumour cluster formation and peritoneal dissemination.Conclusions:Calpain-mediated E-Cad fragmentation appears to promote intraperitoneal EOC progression. Understanding these mechanisms might eventually lead to new tailored subtype-specific diagnostic and therapeutic interventions.

Sexual Activity and Function in Patients With Gynecological Malignancies After Completed Treatment

Objective Sexual activity (SA) and sexual function (SF) after completion of treatment are central for quality of life (QoL) in women affected by gynecological cancer (GC). The aim of this study was to analyze the sexual outcome and overall QoL of women after treatment for primary GC compared with a healthy control group (CG). Methods In a multicenter cross-sectional study, 77 women aged 28 to 67 years were surveyed at least 12 months after completion of primary therapy for cervical, endometrial, or vulvar cancer [gynecological cancer group (GCG)]. Data were collected through validated questionnaires (Female Sexual Function Index-d, EORTC Quality of Life Questionnaire-C30, and Sexual Activity Questionnaire) and compared to a control of 60 healthy women (CG). Results In the GCG, 41.3% were sexually active compared to 78.0% in the CG. Twelve women of the CG and 42 women of the GCG indicated the reasons for their sexual inactivity. The most common reason for sexual inactivity in the GCG was “the-presence-of-a-physical-problem” [18/42 (42.9%) vs 2/12 (16.7%) in the CG], whereas in the CG, “because-I-do-not-have-a-partner” was most common [6/12 (50.0%) vs 11/42 (26.2%) in the GCG]. Sexually active patients in the GCG had an SF comparable to the CG. In multivariate analysis of the total cohort (n = 137), relationship status [solid partnership vs living alone; odds ratio (OR), 33.82; 95% confidence interval (CI), 4.83–236.70], smoking (OR, 0.25; 95% CI, 0.06–1.03), and age (OR, 0.87; 95% CI, 0.79–0.94) influenced SA significantly. The probability of SA thereby decreased with increasing age. Quality of life and subjective general health status were not significantly different between the GCG and the CG (EORTC Quality of Life Questionnaire-C30 score 68.25 vs 69.67). Conclusions A high number of patients with GC remain sexually inactive after treatment, indicating that women experience persistent functional problems. However, women who regain SA after completed treatment have a good overall SF and vice versa.

Prevalence of deleterious germline variants in risk genes including BRCA1/2 in consecutive ovarian cancer patients (AGO-TR-1)

Background Identification of families at risk for ovarian cancer offers the opportunity to consider prophylactic surgery thus reducing ovarian cancer mortality. So far, identification of potentially affected families in Germany was solely performed via family history and numbers of affected family members with breast or ovarian cancer. However, neither the prevalence of deleterious variants in BRCA1/2 in ovarian cancer in Germany nor the reliability of family history as trigger for genetic counselling has ever been evaluated. Methods Prospective counseling and germline testing of consecutive patients with primary diagnosis or with platinum-sensitive relapse of an invasive epithelial ovarian cancer. Testing included 25 candidate and established risk genes. Among these 25 genes, 16 genes (ATM, BRCA1, BRCA2, CDH1, CHEK2, MLH1, MSH2, MSH6, NBN, PMS2, PTEN, PALB2, RAD51C, RAD51D, STK11, TP53) were defined as established cancer risk genes. A positive family history was defined as at least one relative with breast cancer or ovarian cancer or breast cancer in personal history. Results In total, we analyzed 523 patients: 281 patients with primary diagnosis of ovarian cancer and 242 patients with relapsed disease. Median age at primary diagnosis was 58 years (range 16–93) and 406 patients (77.6%) had a high-grade serous ovarian cancer. In total, 27.9% of the patients showed at least one deleterious variant in all 25 investigated genes and 26.4% in the defined 16 risk genes. Deleterious variants were most prevalent in the BRCA1 (15.5%), BRCA2 (5.5%), RAD51C (2.5%) and PALB2 (1.1%) genes. The prevalence of deleterious variants did not differ significantly between patients at primary diagnosis and relapse. The prevalence of deleterious variants in BRCA1/2 (and in all 16 risk genes) in patients <60 years was 30.2% (33.2%) versus 10.6% (18.9%) in patients ≥60 years. Family history was positive in 43% of all patients. Patients with a positive family history had a prevalence of deleterious variants of 31.6% (36.0%) versus 11.4% (17.6%) and histologic subtype of high grade serous ovarian cancer versus other showed a prevalence of deleterious variants of 23.2% (29.1%) and 10.2% (14.8%), respectively. Testing only for BRCA1/2 would miss in our series more than 5% of the patients with a deleterious variant in established risk genes. Conclusions 26.4% of all patients harbor at least one deleterious variant in established risk genes. The threshold of 10% mutation rate which is accepted for reimbursement by health care providers in Germany was observed in all subgroups analyzed and neither age at primary diagnosis nor histo-type or family history sufficiently enough could identify a subgroup not eligible for genetic counselling and testing. Genetic testing should therefore be offered to every patient with invasive epithelial ovarian cancer and limiting testing to BRCA1/2 seems to be not sufficient.

VEGF-C expression attributes the risk for lymphatic metastases to ovarian cancer patients

Background Peritoneal dissemination and retroperitoneal lymph node involvement are main routes for progression of epithelial ovarian cancer (EOC). Vascular endothelial growth factor (VEGF) mediated angiogenesis has been identified as an important mechanism promoting tumour progression. Methods Tumour tissue of 100 patients with EOC was analysed for protein expression of vascular endothelial growth factor (VEGF)-A, -C, -D by Western Blot analysis. Expression patterns in patients with ‘extensive intraperitoneal’ metastases (pT3c pN1 and pT3b-pT3c pN0, n=80) were compared to patients with ‘predominantly retroperitoneal’ metastases (pT1a-pT3b, pN1, n=20). Overall and progression-free survival was analysed by Kaplan-Meier method. Results While no significant differences in expression levels among the different modes of metastases were noted for VEGF-A and -D, VEGF-C expression was significantly higher in the group of predominantly retroperitoneal metastases compared to the group with extensive intraperitoneal metastases. Patients with high VEGF-C expression had a significantly worse overall survival compared to patients with low expression levels. Conclusions Retroperitoneal tumour progression in EOC patients is associated with high VEGF-C expression. VEGF-C may serve as a molecular marker to identify patients with assumed high risk for lymphatic metastases, who might benefit from specific treatment strategies.

Beyond Bevacizumab: An Outlook to New Anti-Angiogenics for the Treatment of Ovarian Cancer

In addition to the monoclonal vascular endothelial growth factor (VEGF) antibody bevacizumab, several alternative anti-angiogenic treatment strategies for ovarian cancer patients have been evaluated in clinical trials. Apart from targeting extracellular receptors by the antibody aflibercept or the peptibody trebananib, the multikinase inhibitors pazopanib, nintedanib, cediranib, sunitinib, and sorafenib were developed to interfere with VEGF receptors and multiple additional intracellular pathways. Nintedanib and pazopanib significantly improved progression-free survival in two positive phase III trials for first-line therapy. A reliable effect on overall survival could, however, not be observed for any anti-angiogenic first-line therapies so far. In terms of recurrent disease, two positive phase III trials revealed that trebananib and cediranib are effective anti-angiogenic agents for this indication. Patient selection and biomarker guided prediction of response seems to be a central aspect for future studies. Combining anti-angiogenics with other targeted therapies to possibly spare chemotherapy in certain constellations represents another very interesting future perspective for clinical trials. This short review gives an overview of current clinical trials for anti-angiogenic treatment strategies beyond bevacizumab. In this context, possible future perspectives combining anti-angiogenics with other targeted therapies and the need for specific biomarkers predicting response are elucidated.

Loss of BRCA1 promotor hypermethylation in recurrent high-grade ovarian cancer

Background Approximately 20-25% of ovarian cancers are attributable to germline or somatic BRCA1/2 mutations, resulting in defects in the homologous recombination pathway. Inactivation of these genes can also be mediated by epigenetic changes, e.g., hypermethylation of CpG islands in the promoter regions. In such homologous recombination deficient tumors, platinum based chemotherapy is in general effective, however, loss of hypermethylation might lead to refractory disease. The aim of this study was to evaluate the stability of BRCA1 promoter hypermethylation in recurrent disease after platinum based chemotherapy. Methods Tumor tissue from 76 patients with primary and 48 patients with platinum-sensitive recurrent high-grade ovarian cancer was collected. In a subgroup of 12 patients, ‘paired’ tumor tissue from primary and recurrent surgery was available. BRCA1 promoter methylation status was assessed using methylation specific polymerase chain reaction and was verified by Sanger Sequencing. Results 73.7% (56/76) of primary and 20.8% (10/48) of recurrent tumors displayed BRCA1 promoter hypermethylation. BRCA1 promoter methylation status was not associated with progression-free- or overall survival. In the paired subgroup 83.3% (10/12) of the primary vs. 16.7% (2/12) of the recurrent tumors showed hypermethylation. In eight patients loss of BRCA1 hypermethylation was observed, whereas two patients had stable methylation status. Conclusions Loss of BRCA1 promoter methylation may be a mechanism to restore BRCA1 function in recurrent disease. However, currently the clinical significance is still unclear and should be evaluated in prospective clinical trials.

Abstract 2421: Circulating tumor cells reveal the genetic evolution of metastatic breast cancer

BACKGROUND. Anti-cancer treatment failure is caused by the genetic and phenotypic heterogeneous properties of the disease. Early dissemination of circulating tumor cells (CTCs) into the blood circulation of cancer patients allows for parallel genetic evolution of the primary tumor and metastases. This study investigated the genomic make-up of CTCs originating from metastasis as a so called “liquid biopsy” and compared these with the different clones of the archived autologous primary tumor on single cell level. METHODS. From two breast cancer patients, individual CTCs were isolated from blood using Ficoll density gradient followed by micromanipulation of keratin positive cells. Single cells from archived primary breast tumors from the same patients were captured by laser microdissection. DNA was isolated and amplified by whole genome amplification and copy number alterations (CNA) were obtained by shallow, whole genome next generations sequencing (NGS). RESULTS. From the first breast cancer patient, the genomes from 50 single cells from the primary tumor were sequenced. An unsupervised phylogenetic cluster analysis based on CNA revealed five distinct subclones that displayed increasing chromosomal instability from the first to the last cluster. Using support vector machine (SVM) learning, the CNA profiles of 42 CTCs were residing mostly to the first three clones with few chromosomal aberrations, whereas only one CTC resided to the fourth clone and none to the last with many chromosomal aberrations. The tumor from a second breast cancer patient displayed the presence of three genetically distinct clones with increasing chromosomal instability after sequencing 11 single cells. CTCs (n = 12) from this patient at metastatic disease were classified to the last branch using SVM, however with low probability. After repeating unsupervised clustering on the genomes of the primary tumor tissue and CTCs, a fourth branch was formed with CTCs only. CONCLUSION. Our results suggest that therapy resistant metastases in breast cancer patients can originate from tumor clones from early stages of tumor evolution and may genetically still be similar (patient 1). On the other hand, further genetic progression may also take place (patient 2) after which the genetic landscape of the metastasis does not resemble the primary tumor anymore. These results underline the importance of “liquid biopsy” in the diagnosis of metastatic cancer. Citation Format: Simon A. Joosse, Anna Babayan, Katharina Prieske, Daniela Indenbirken, Malik Alawi, Eike C. Burandt, Adam Grundhoff, Volkmar Muller, Klaus Pantel. Circulating tumor cells reveal the genetic evolution of metastatic breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2421.

Diagnosis of Li-Fraumeni Syndrome: Differentiating TP53 germline mutations from clonal hematopoiesis: Results of the observational AGO-TR1 trial

The Li‐Fraumeni cancer predisposition syndrome (LFS1) presents with a variety of tumor types and the TP53 gene is covered by most diagnostic cancer gene panels. We demonstrate that deleterious TP53 variants identified in blood‐derived DNA of 523 patients with ovarian cancer (AGO‐TR1 trial) were not causal for the patients’ ovarian cancer in three out of six TP53‐positive cases. In three out of six patients, deleterious TP53 mutations were identified with low variant fractions in blood‐derived DNA but not in the tumor of the patient seeking advice. The analysis of the TP53 and PPM1D genes, both intimately involved in chemotherapy‐induced and/or age‐related clonal hematopoiesis (CH), in 523 patients and 1,053 age‐matched female control individuals revealed that CH represents a frequent event following chemotherapy, affecting 26 of the 523 patients enrolled (5.0%). Considering that TP53 mutations may arise from chemotherapy‐induced CH, our findings help to avoid false‐positive genetic diagnoses of LFS1.

Participation of elderly gynecological cancer patients in clinical trials

BackgroundElderly patients are underrepresented in clinical trials in gynecological cancer, even though they are disproportionally often affected. This study aimed to evaluate the disposition and apprehension of elderly patients toward study participation.Methods112 elderly gynecological cancer patients (median age 70) were surveyed in a multicenter cross-sectional study. Besides fitness, state of disease, education and domestic situation, questions aimed at the general willingness to participate in a clinical trial. Personal reasons for refusal and anticipated advantages/disadvantages that might evolve from participation were inquired.ResultsWillingness to participate in a clinical study was generally high (72%, 74/102). Reasons for potential study participation were: ‘better monitoring of the disease’ (67.1%), ‘better medical care’ (46.1%), ‘to help medical research’ (44.7%), ‘better medication’ (35.5%) and ‘because of my doctor’s recommendation’ (22.4%). Reasons for potential refusal were: ‘too time consuming’ (24.4%), ‘fear of side effects’ (21.8%), ‘misuse as experimental animal’ (18%), ‘long distance to clinic’ (14.1%) and ‘too little or unclear information’ (10.3%). 37.2% (29/78) of the patients stated that they had ‘no objection’ at all against study participation. The question if patients anticipated having a longer life due to study participation was answered with ‘yes’ or ‘rather yes’ in 42% (38/90); 28.9% answered ‘no’ or ‘rather no’ (29% undecided). No statistical significant relation between willingness to participate in a study and general fitness (p = 0.133), education (p = 0.122), age (p = 0.474) or domestic situation (p = 0.123) could be observed in a multivariate logistic regression model.ConclusionsElderly patients are generally willing to participate in clinical studies, in our cohort regardless of their fitness. Benefits of participation seem to be unclear among a majority of potential study participants. Therefore, it might be decisive to provide more general information regarding benefits and safety for elderly patients in a clinical trial.