Guenter Niegisch

Time from Prior Chemotherapy Enhances Prognostic Risk Grouping in the Second-line Setting of Advanced Urothelial Carcinoma: A Retrospective Analysis of Pooled, Prospective Phase 2 Trials

BACKGROUND Outcomes for patients in the second-line setting of advanced urothelial carcinoma (UC) are dismal. The recognized prognostic factors in this context are Eastern Cooperative Oncology Group (ECOG) performance status (PS) >0, hemoglobin level (Hb) <10 g/dl, and liver metastasis (LM). OBJECTIVES The purpose of this retrospective study of prospective trials was to investigate the prognostic value of time from prior chemotherapy (TFPC) independent of known prognostic factors. DESIGN, SETTING, AND PARTICIPANTS Data from patients from seven prospective trials with available baseline TFPC, Hb, PS, and LM values were used for retrospective analysis (n=570). External validation was conducted in a second-line phase 3 trial comparing best supportive care (BSC) versus vinflunine plus BSC (n=352). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Cox proportional hazards regression was used to evaluate the association of factors, with overall survival (OS) and progression-free survival (PFS) being the respective primary and secondary outcome measures. RESULTS AND LIMITATIONS ECOG-PS >0, LM, Hb <10 g/dl, and shorter TFPC were significant prognostic factors for OS and PFS on multivariable analysis. Patients with zero, one, two, and three to four factors demonstrated median OS of 12.2, 6.7, 5.1, and 3.0 mo, respectively (concordance statistic=0.638). Setting of prior chemotherapy (metastatic disease vs perioperative) and prior platinum agent (cisplatin or carboplatin) were not prognostic factors. External validation demonstrated a significant association of TFPC with PFS on univariable and most multivariable analyses, and with OS on univariable analyses. Limitations of retrospective analyses are applicable. CONCLUSIONS Shorter TFPC enhances prognostic classification independent of ECOG-PS >0, Hb <10 g/dl, and LM in the setting of second-line therapy for advanced UC. These data may facilitate drug development and interpretation of trials.

Single-agent Taxane Versus Taxane-containing Combination Chemotherapy as Salvage Therapy for Advanced Urothelial Carcinoma

BACKGROUND Single-agent taxanes are commonly used as salvage systemic therapy for patients with advanced urothelial carcinoma (UC). OBJECTIVE To study the impact of combination chemotherapy delivering a taxane plus other chemotherapeutic agents compared with single-agent taxane as salvage therapy. DESIGN, SETTING, AND PARTICIPANTS Individual patient-level data from phase 2 trials of salvage systemic therapy were used. INTERVENTIONS Trials evaluating either single agents (paclitaxel or docetaxel) or combination chemotherapy (taxane plus one other chemotherapeutic agent or more) following prior platinum-based therapy were used. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Information regarding the known major baseline prognostic factors was required: time from prior chemotherapy, hemoglobin, performance status, albumin, and liver metastasis status. Cox proportional hazards regression was used to evaluate the association of prognostic factors and combination versus single-agent chemotherapy with overall survival (OS). RESULTS AND LIMITATIONS Data were available from eight trials including 370 patients; two trials (n=109) evaluated single-agent chemotherapy with docetaxel (n=72) and cremophor-free paclitaxel (n=37), and six trials (n=261) evaluated combination chemotherapy with gemcitabine-paclitaxel (two trials, with n=99 and n=24), paclitaxel-cyclophosphamide (n=32), paclitaxel-ifosfamide-nedaplatin (n=45), docetaxel-ifosfamide-cisplatin (n=26), and paclitaxel-epirubicin (n=35). On multivariable analysis after adjustment for baseline prognostic factors, combination chemotherapy was independently and significantly associated with improved OS (hazard ratio: 0.60; 95% confidence interval, 0.45-0.82; p=0.001). The retrospective design of this analysis and the trial-eligible population were inherent limitations. CONCLUSIONS Patients enrolled in trials of combination chemotherapy exhibited improved OS compared with patients enrolled in trials of single-agent chemotherapy as salvage therapy for advanced UC. Prospective randomized trials are required to validate a potential role for rational and tolerable combination chemotherapeutic regimens for the salvage therapy of advanced UC. PATIENT SUMMARY This retrospective study suggests that a combination of chemotherapy agents may extend survival compared with single-agent chemotherapy in selected patients with metastatic urothelial cancer progressing after prior chemotherapy.

Six-month progression-free survival as the primary endpoint to evaluate the activity of new agents as second-line therapy for advanced urothelial carcinoma.

OBJECTIVE Second-line systemic therapy for advanced urothelial carcinoma (UC) has substantial unmet needs, and current agents show dismal activity. Second-line trials of metastatic UC have used response rate (RR) and median progression-free survival (PFS) as primary endpoints, which may not reflect durable benefits. A more robust endpoint to identify signals of durable benefits when investigating new agents in second-line trials may expedite drug development. PFS at 6 months (PFS6) is a candidate endpoint, which may correlate with overall survival (OS) at 12 months (OS12) and may be applicable across cytostatic and cytotoxic agents. METHODS Ten second-line phase II trials with individual patient outcomes data evaluating chemotherapy or biologics were combined for discovery, followed by external validation in a phase III trial. The relationship between PFS6/RR and OS12 was assessed at the trial level using Pearson correlation and weighted linear regression, and at the individual level using Pearson chi-square test with Yates continuity correction. RESULTS In the discovery dataset, a significant correlation was observed between PFS6 and OS12 at the trial (R(2) = 0.55, Pearson correlation = 0.66) and individual levels (82%, Қ = 0.45). Response correlated with OS12 at the individual level less robustly (78%, Қ = 0.36), and the trial level association was not statistically significant (R(2) = 0.16, Pearson correlation = 0.37). The correlation of PFS6 (81%, Қ = 0.44) appeared stronger than the correlation of response (76%, Қ = 0.17) with OS12 in the external validation dataset. CONCLUSIONS PFS6 is strongly associated with OS12 and appears more optimal than RR to identify active second-line agents for advanced UC.

Pathological T0 Following Radical Cystectomy with or without Neoadjuvant Chemotherapy: A Useful Surrogate

PURPOSE Several large, randomized, controlled trials provide evidence that neoadjuvant chemotherapy improves the outcome of radical cystectomy for muscle invasive urothelial bladder cancer. We analyzed the designs, methods and observations of these trials to identify patient subgroups that appeared most likely to benefit. We also identified distinguishing features compared to groups that did not achieve improved outcomes. MATERIALS AND METHODS We analyzed initial and updated methods and results of the 4 main prospective trials of neoadjuvant chemotherapy (SWOG, Medical Research Council, and Nordic I and II) and subsequent meta-analyses. These series are the basis for advocating neoadjuvant chemotherapy in all patients with muscle invasive urothelial bladder cancer who undergo radical cystectomy. RESULTS The greatest apparent benefit was seen in patients free of cancer at radical cystectomy (pT0). They had markedly improved overall and disease specific survival compared to patients with residual disease. However, improvements occurred regardless of whether there was down-staging from muscle invasive urothelial bladder cancer to pT0 after transurethral resection alone (controls) or after resection plus neoadjuvant chemotherapy. Thus, the major benefit of chemotherapy appeared to be that more patients achieved pT0. We also explored the study limitations that may have influenced outcomes and considered the potential for overtreatment in patients not likely to benefit from chemotherapy. Finally, we used risk stratification to create a decision tree model for selecting patients for neoadjuvant chemotherapy that could conceivably maximize oncologic outcome and minimize overtreatment. CONCLUSIONS Patients with pT0 in the 4 main neoadjuvant chemotherapy trials and their subsequent meta-analyses experienced similar survival, far exceeding that in groups that did not achieve pT0. The benefit of neoadjuvant chemotherapy appears to be the larger number of cases than in the transurethral resection only group that were down-staged to pT0, suggesting that variables other than chemotherapy may have influenced outcomes. Therefore, strategies to selectively administer neoadjuvant chemotherapy to certain patients at risk have the potential to maintain improved bladder cancer outcomes while reducing overtreatment and its associated toxicity.

Impact of Response to Prior Chemotherapy in Patients With Advanced Urothelial Carcinoma Receiving Second-Line Therapy: Implications for Trial Design

BACKGROUND The prognostic impact of response to prior chemotherapy independent of performance status (PS), hemoglobin (Hb), liver metastasis (LM), and time from prior chemotherapy (TFPC) in the context of second-line therapy for advanced urothelial carcinoma (UC) is unknown. METHODS Six phase II trials evaluating second-line therapy (n = 504) were pooled. Patients who received prior therapy for metastatic disease were eligible for analysis if Hb, LM, PS, and TFPC were available. Response by Response Evaluation Criteria in Solid Tumors 1.0 to first-line therapy was recorded. Progression-free survival (PFS) and overall survival (OS) were calculated from the date of registration using the Kaplan-Meier method. RESULTS A total of 275 patients were evaluable for analysis. Patients received gemcitabine-paclitaxel, cyclophosphamide-paclitaxel, pazopanib, docetaxel plus vandetanib/placebo, or vinflunine (2 trials). Those with prior response (n = 111) had a median OS of 8.0 months (95% confidence interval [CI], 6.8-9.4), compared with 5.9 months (95% CI, 5.0-6.6) for those without prior response (n = 164). Those with prior response had a median PFS of 3.0 months (95% CI, 2.6-4.0) compared with 2.6 months (95% CI, 2.0-2.8) in patients without response. Multivariable analysis did not reveal a significant independent impact of prior response on PFS and OS. CONCLUSIONS Best prior response in patients receiving prior chemotherapy for metastatic disease did not confer an independent prognostic impact with second-line therapy for advanced UC. Given that the setting of prior chemotherapy (metastatic or perioperative) has not appeared significant in a prior study, patients who received prior chemotherapy in perioperative or metastatic settings may be enrolled in the same second-line trial stratified for PS, Hb, LM, and TFPC.

Inhibition of class I histone deacetylases 1 and 2 promotes urothelial carcinoma cell death by various mechanisms

Class I histone deacetylases HDAC1 and HDAC2 contribute to cell proliferation and are commonly upregulated in urothelial carcinoma. To evaluate whether specific inhibition of these enzymes might serve as an appropriate therapy for urothelial carcinoma, siRNA-mediated knockdown and specific pharmacologic inhibition of HDAC1 and HDAC2 were applied in urothelial carcinoma cell lines (UCC) with distinct HDAC1 and HDAC2 expression profiles. HDACs and response marker proteins were followed by Western blotting and qRT-PCR. Effects of class I HDAC suppression on UCCs were analyzed by viability, colony forming, and caspase-3/7 assays; flow cytometry, senescence and lactate dehydrogenase cytotoxicity assays; and immunofluorescence staining. Whereas single knockdowns of HDAC1 or HDAC2 were impeded by compensatory upregulation of the other isoenzyme, efficient double knockdown of HDAC1 and HDAC2 reduced proliferation by up to 80% and induced apoptosis-like cell death in all UCCs. Clonogenic growth was cell line– and HDAC-dependently reduced, with double knockdown of HDAC1 and HDAC2 being usually most efficient. Class I HDAC-specific inhibitors, especially the more specific HDAC1/2 inhibitors romidepsin and givinostat, significantly reduced proliferation of all UCCs (IC50, 3.36 nmol/L–4.59 μmol/L). Romidepsin and givinostat also significantly inhibited clonogenic growth of UCCs, with minor effects on nontumorigenic controls. Intriguingly, these compounds induced primarily S-phase disturbances and nonapoptotic cell death in UCCs. Thus, although both ways of inhibiting HDAC1/2 share mechanisms and efficaciously inhibit cell proliferation, their modes of action differ substantially. Regardless, combined inhibition of HDAC1/2 appears to represent a promising strategy for urothelial carcinoma therapy. Mol Cancer Ther; 15(2); 299–312. ©2016 AACR.

Phenotype plasticity rather than repopulation from CD90/CK14+ cancer stem cells leads to cisplatin resistance of urothelial carcinoma cell lines

BackgroundTumour heterogeneity and resistance to systemic treatment in urothelial carcinoma (UC) may arise from cancer stem cells (CSC). A recent model describes cellular differentiation states within UC based on corresponding expression of surface markers (CD) and cytokeratins (CK) with CD90 and CK14 positive cells representing the least differentiated and most tumourigenic population. Based on the fact that this population is postulated to constitute CSCs and the origin of cisplatin resistance, we enriched urothelial carcinoma cell lines (UCCs) for CD90 and studied the tumour-initiating potential of these separated cells in vitro.MethodsMagnetic- and fluorescence-activated- cell sorting were used for separation of CD90+ and CD90− UCCs. Distribution of cell surface markers CD90, CD44, and CD49f and cytokeratins CK14, CK5, and CK20 as well as the effects of short- and long-term treatment with cisplatin were assessed in vitro and measured by qRT-PCR, immunocytochemistry, reporter assay and flow cytometry in 11 UCCs.ResultsWe observed cell populations with surface markers according to those reported in tumour xenografts. However, expression of cytokeratins did not concord regularly with that of the surface markers. In particular, expression of CD90 and CK14 diverged during enrichment of CD90+ cells by immunomagnetic sorting or following cisplatin treatment. Enriched CD90+ cells did not exhibit CSC-like characteristics like enhanced clonogenicity and cisplatin resistance. Moreover, selection of cisplatin-resistant sublines by long-term drug treatment did not result in enrichment of CD90+ cells. Rather, these sublines displayed significant phenotypic plasticity expressing EMT markers, an altered pattern of CKs, and WNT-pathway target genes.ConclusionsOur findings indicate that the correspondence between CD surface markers and cytokeratins reported in xenografts is not maintained in commonly used UCCs and that CD90 may not be a stable marker of CSC in UC. Moreover, UCCs cells are capable of substantial phenotypic plasticity that may significantly contribute to the emergence of cisplatin resistance.

Cisplatin-Based First-Line Therapy for Advanced Urothelial Carcinoma After Previous Perioperative Cisplatin-Based Therapy

BACKGROUND Outcomes with cisplatin-based first-line therapy for advanced UC after previous perioperative cisplatin-based chemotherapy are unclear. In this study we evaluated outcomes with a focus on the effect of time from previous cisplatin-based perioperative chemotherapy. PATIENTS AND METHODS Data were collected for patients who received cisplatin-based first-line therapy for advanced UC after previous perioperative cisplatin-based therapy. Cox proportional hazards models were used to investigate the prognostic ability of visceral metastasis, ECOG PS, TFPC, anemia, leukocytosis, and albumin on overall survival (OS). RESULTS Data were available for 41 patients from 8 institutions including 31 men (75.6%). The median age was 61 (range, 41-77) years, most received gemcitabine plus cisplatin (n = 26; 63.4%), and the median number of cycles was 4 (range, 1-8). The median OS was 68 weeks (95% confidence interval [CI], 48.0-81.0). Multivariable Cox regression analysis results showed an independent prognostic effect on OS for PS > 0 versus 0 (hazard ratio [HR], 4.56 [95% CI, 1.66-12.52]; P = .003) and TFPC ≥ 78 weeks versus < 78 weeks (HR, 0.48 [95% CI, 0.21-1.07]; P = .072). The prognostic model for OS was internally validated with c-index = 0.68. Patients with TFPC < 52 weeks, 52 to 104 weeks, and ≥ 104 weeks had median survival of 42, 70, and 162 weeks, respectively. CONCLUSION Longer TFPC ≥ 78 weeks and ECOG PS = 0 were independently prognostic for better survival with cisplatin-based first-line chemotherapy for advanced UC after previous perioperative cisplatin-based chemotherapy. The data support using TFPC ≥ 52 weeks to rechallenge with cisplatin-based first-line chemotherapy for metastatic disease.

Venous thromboembolism in metastatic urothelial carcinoma or variant histologies: incidence, associative factors, and effect on survival

Venous thromboembolism (VTE) is common in cancer patients. However, little is known about VTE risk in metastatic urothelial carcinoma or variant histologies (UC/VH). We sought to characterize the incidence, associative factors, including whether various chemotherapy regimens portend different risk, and impact of VTE on survival in metastatic UC/VH patients. Patients diagnosed with metastatic UC/VH from 2000 to 2013 were included in this multicenter retrospective, international study from 29 academic institutions. Cumulative and 6‐month VTE incidence rates were determined. The association of first‐line chemotherapy (divided into six groups) and other baseline characteristics on VTE were analyzed. Each chemotherapy treatment group and statistically significant baseline clinical characteristics were assessed in a multivariate, competing‐risk regression model. VTE patients were matched to non‐VTE patients to determine the impact of VTE on overall survival. In all, 1762 patients were eligible for analysis. There were 144 (8.2%) and 90 (5.1%) events cumulative and within the first 6 months, respectively. VTE rates based on chemotherapy group demonstrated no statistical difference when gemcitabine/cisplatin was used as the comparator. Non‐urotheilal histology (SHR: 2.67; 95% CI: 1.72–4.16, P < 0.001), moderate to severe renal dysfunction (SHR: 2.12; 95% CI: 1.26–3.59, P = 0.005), and cardiovascular disease (CVD) or CVD risk factors (SHR: 2.27; 95% CI: 1.49–3.45, P = 0.001) were associated with increased VTE rates. Overall survival was worse in patients with VTE (median 6.0 m vs. 10.2 m, P < 0.001). Thus, in metastatic UC/VH patients, VTE is common and has a negative impact on survival. We identified multiple associated potential risk factors, although different chemotherapy regimens did not alter risk.

Applying the chicken embryo chorioallantoic membrane assay to study treatment approaches in urothelial carcinoma

BACKGROUND Rapid development of novel treatment options demands valid preclinical screening models for urothelial carcinoma (UC). The translational value of high-throughput drug testing using 2-dimensional (2D) cultures is limited while for xenograft models handling efforts and costs often become prohibitive for larger-scale drug testing. Therefore, we investigated to which extent the chicken chorioallantoic membrane (CAM) assay might provide an alternative model to study antineoplastic treatment approaches for UC. METHODS The ability of 8 human UC cell lines (UCCs) to form tumors after implantation on CAMs was investigated. Epithelial-like RT-112 and mesenchymal-like T-24 UCCs in cell culture or as CAM tumors were treated with cisplatin alone or combined with histone deacetylase inhibitors (HDACi) romidepsin and suberanilohydroxamic acid. Tumor weight, size, and bioluminescence activity were monitored; tumor specimens were analyzed by histology and immunohistochemistry. Western blotting and quantitative real time polymerase chain reaction were used to measure protein and mRNA expression. RESULTS UCCs were reliably implantable on the CAM, but tumor development varied among cell lines. Expression of differentiation markers (E-cadherin, vimentin, CK5, CK18, and CK20) was similar in CAM tumors and 2D cultures. Cellular phenotypes also remained stable after recultivation of CAM tumors in 2D cultures. Bioluminescence images correlated with tumor weight. Cisplatin and HDACi decreased weight and growth of CAM tumors in a dose-dependent manner, but HDACi treatment acted less efficiently as in 2D cultures, especially on its typically associated molecular markers. Synergistic effects of HDACi and subsequent cisplatin treatment on UCCs were neither detected in 2D cultures nor detected in CAM tumors. CONCLUSION Our results demonstrate that the CAM assay is a useful tool for studying tumor growth and response to conventional anticancer drugs under 3D conditions, especially cytotoxic drugs as cisplatin. With some limitations, it might serve as a cost- and time-effective preclinical screening assay for novel therapeutic approaches before further assessment in expensive and cumbersome animal models.