David J. McConkey

Predicting Response to Intravesical Bacillus Calmette-Guérin Immunotherapy: Are We There Yet? A Systematic Review

CONTEXT Bacillus Calmette-Guérin (BCG) is currently the most effective intravesical therapy for nonmuscle invasive bladder cancer, reducing not only recurrence rates but also preventing progression and reducing deaths. However, response rates to BCG vary widely and are dependent on a multitude of factors. OBJECTIVE To review existing data on clinical, pathologic, immune, and molecular markers that allow prediction of BCG response. EVIDENCE ACQUISITION PubMed and MEDLINE search of English language literature was conducted from its inception to July 2017 using appropriate search terms. Following systematic literature review and analysis of data, consensus voting was used to generate the content of this review. EVIDENCE SYNTHESIS As seen in the EORTC and CUETO risk nomograms, clinicopathologic features, especially tumor stage and grade, are the most effective predictors of BCG response. Data are less robust with regards to the association of response with age, female sex, recurrent tumors, multiplicity of tumors, and the presence of carcinoma in situ. Single biomarkers, such as tumor p53 and urinary interleukin-2 expression, have had limited success in predicting BCG response, possibly due to the multifaceted nature of the generated immune response. More comprehensive biomarker panels (eg, urinary cytokines), have a more robust correlation with response, as do patterns of urinary cytologic fluorescent in-situ hybridization examination. Gene expression data correlate with disease progression, but studies examining potential associations with BCG response are limited. CONCLUSIONS Currently, the best predictors of BCG response are clinicopathologic features such as tumor grade and stage. Panels of urinary cytokines, as well as fluorescent in-situ hybridization patterns of cytologic anomalies, appear to be promising biomarkers. The complexity of the immune response to BCG and the heterogeneity of bladder cancer suggest that future studies should amalgamate measures reflecting innate immune response and tumor/stromal gene expression before these can be adopted for clinical use. PATIENT SUMMARY Bacillus Calmette-Guérin (BCG) immunotherapy is an effective treatment for many patients with nonmuscle invasive bladder cancer. An individuals response to BCG can be predicted by using various features of the cancer. In the future, predictive markers using molecular measures of the tumor type and the immune response to BCG may allow us to precisely know an individuals likely outcome after BCG treatment.

The integrated stress response and proteotoxicity in cancer therapy

A variety of different forms of cellular stress can cause protein misfolding and aggregation and proteotoxicity. The cytoprotective response to proteotoxicity is termed the integrated stress response and involves 4 distinct serine/threonine protein kinases that converge on the translation initiation factor eIF2α, resulting in phosphorylation at S51, cell cycle arrest, and a general inhibition of global protein synthesis. Phosphorylation of eIF2α also promotes translation of ATF4 and the expression of ATF4 target genes that ameliorate proteotoxic stress but can also promote apoptosis. This mini review provides a general overview of these mechanisms and discusses how the inter-tumor heterogeneity that involves them affects sensitivity and resistance to proteasome inhibitors, a new class of cancer therapeutics that promotes tumor cell killing via proteotoxic stress.

Specific micro-RNA expression patterns distinguish the basal and luminal subtypes of muscle-invasive bladder cancer.

The roles of non-coding RNAs in controlling clinical and biological heterogeneity in bladder cancer remain unclear. We used TCGAs published dataset (n = 405 tumors) as a discovery cohort and created a new validation cohort to define the miRNA expression patterns in the basal and luminal molecular subtypes of muscle-invasive bladder cancer (MIBC). We identified 63 miRNAs by PAM, which optimally identified basal and luminal tumors. The targets of the top luminal miRNAs were activators of EMT (ZEB1, ZEB2) and basal subtype transcription (IL-6, EGFR, STAT3), whereas the targets of the top basal miRNAs were involved in adipogenesis pathways and luminal breast cancer (ERBB2, ERBB3). We also identified a 15-miRNA signature that identified stromally infiltrated basal and luminal MIBCs corresponding to the “cluster IV/immune undifferentiated/claudin-low” and “cluster II/luminal immune” subtypes identified previously, which likely contain samples with higher infiltration rates. Using the 63-miRNA signature, we accurately assigned MIBCs to the basal and luminal subtypes and confirmed that patients with basal tumors had shorter overall survival. The results strongly suggest that miRNAs contribute to the control of the gene expression patterns observed in basal and luminal MIBCs and that they can be used as biomarkers and candidate therapeutic targets.

Intravesical BCG Induces CD4+ T-cell expansion in an immune competent model of bladder cancer

Intravesical bacillus Calmette–Guéerin (BCG) instillations are standard of care for early stage bladder cancer. BCG was found to recruit T cells to the bladder, but their phenotype was unchanged, implying that combining T cell–activating agents with BCG might improve clinical activity. Intravesical bacillus Calmette–Guérin (BCG) immunotherapy is the standard of care in treating non–muscle-invasive bladder cancer, yet its mechanism of action remains elusive. Both innate and adaptive immune responses have been implicated in BCG activity. Although prior research has indirectly demonstrated the importance of T cells and shown a rise in CD4+ T cells in bladder tissue after BCG, T-cell subpopulations have not been fully characterized. We investigated the relationship between effector and regulatory T cells in an immune competent, clinically relevant rodent model of bladder cancer. Our data demonstrate that cancer progression in the N-methyl-N-nitrosourea (MNU) rat model of bladder cancer was characterized by a decline in the CD8/FoxP3 ratio, consistent with decreased adaptive immunity. In contrast, treatment with intravesical BCG led to a large, transient rise in the CD4+ T-cell population in the urothelium and was both more effective and immunogenic compared with intravesical chemotherapy. Whole-transcriptome expression profiling of posttreatment intravesical CD4+ and CD8+ T cells revealed minimal differences in gene expression after BCG treatment. Together, our results suggest that although BCG induces T-cell recruitment to the bladder, the T-cell phenotype does not markedly change, implying that combining T-cell–activating agents with BCG might improve clinical activity. Cancer Immunol Res; 5(7); 594–603. ©2017 AACR.

Intrinsic subtypes and bladder cancer metastasis

Recent studies demonstrated that bladder cancers can be grouped into basal and luminal molecular subtypes that possess distinct biological and clinical characteristics. Basal bladder cancers express biomarkers characteristic of cancer stem cells and epithelial-to-mesenchymal transition (EMT). Patients with basal cancers tend have more advanced stage and metastatic disease at presentation. In preclinical models basal human orthotopic xenografts are also more metastatic than luminal xenografts are, and they metastasize via an EMT-dependent mechanism. However, preclinical and clinical data suggest that basal cancers are also more sensitive to neoadjuvant chemotherapy (NAC), such that most patients with basal cancers who are aggressively managed with NAC have excellent outcomes. Importantly, luminal bladder cancers can also progress to become invasive and metastatic, but they appear to do so via mechanisms that are much less dependent on EMT and may involve help from stromal cells, particularly cancer-associated fibroblasts (CAFs). Although patients with luminal cancers do not appear to derive much clinical benefit from NAC, the luminal tumors that are infiltrated with stromal cells appear to be sensitive to anti-PDL1 antibodies and possibly other immune checkpoint inhibitors. Therefore, neoadjuvant and/or adjuvant immunotherapy may be the most effective approach in treating patients with advanced or metastatic infiltrated luminal bladder cancers.

Preclinical Evaluation of Intravesical Cisplatin Nanoparticles for Non-Muscle-Invasive Bladder Cancer

Purpose: Prior clinical trials evaluating cisplatin for non–muscle-invasive bladder cancer (NMIBC) were stopped due to local and systemic toxicity. Currently, there is still a need for improved intravesical therapies, and nanoparticle-based CDDP may be efficacious without the toxicity of free cisplatin observed in the past. Experimental Design: Cisplatin nanoparticles (CDDP NPs) were developed using biocompatible poly(l-aspartic acid sodium salt; PAA), both with and without low and high grafting density of methoxy-polyethylene glycol (PEG). In vitro cytotoxicity studies confirmed activity of CDDP NPs and CDDP solution against a papillary bladder cancer cell line. Local toxicity was assessed by three weekly intravesical administrations of CDDP formulations. CDDP NPs and CDDP solution were evaluated for bladder absorption in murine models 1 and 4 hours after intravesical administration. In vivo efficacy was evaluated in an immunocompetent carcinogen model of NMIBC. Results: CDDP NPs showed decreased local toxicity, as assessed by bladder weight, compared with CDDP solution. Furthermore, >2 μg/mL of platinum was observed in mouse serum after intravesical administration of CDDP solution, whereas serum platinum was below the limit of quantification after intravesical administration of CDDP NPs. CDDP NPs provided significantly increased (P < 0.05) drug levels in murine bladders compared with CDDP solution for at least 4 hours after intravesical administration. In vivo, CDDP NPs reduced cancer cell proliferation compared with untreated controls, and was the only treatment group without evidence of invasive carcinoma. Conclusions: Cisplatin-loaded PAA NPs have the potential to improve intravesical treatment of NMIBC while reducing local and systemic side effects. Clin Cancer Res; 23(21); 6592–601. ©2017 AACR.

Comparison of Pathological Stage in Patients Treated with and without Neoadjuvant Chemotherapy for High Risk Upper Tract Urothelial Carcinoma

Purpose: High risk upper tract urothelial carcinoma has been associated with poor survival outcomes. Limited retrospective data support neoadjuvant chemotherapy prior to radical nephroureterectomy. To validate prior findings we evaluated differences in the pathological stage distribution in patients with high risk upper tract urothelial carcinoma based on the administration of neoadjuvant chemotherapy before radical nephroureterectomy. Materials and Methods: We retrospectively analyzed the records of 240 patients with upper tract urothelial carcinoma at The Johns Hopkins Hospital from 2003 to 2017. Patients with biopsy proven high grade disease and a visible lesion on cross‐sectional imaging were offered neoadjuvant chemotherapy prior to radical nephroureterectomy. A control group of a time matched cohort of patients with biopsy proven high grade disease underwent extirpative surgery alone. The chi‐square and Fisher exact tests were used to evaluate clinical and pathological variables between the cohorts. Results: There were 32 patients in the study group and 208 in the control group. Significantly lower pathological stage was noted in the study group than in the control group (p <0.001). Significantly fewer patients with pT2 disease or higher were treated with neoadjuvant chemotherapy (37.5% vs 59.6%, p = 0.02). There was a 46.5% reduction in the prevalence of pT3 disease or higher in study group patients without clinically node positive or low volume metastatic disease (25.9% vs 48.4%, p = 0.04). A 9.4% complete remission rate was observed in patients who underwent neoadjuvant chemotherapy. Conclusions: Patients with high risk upper tract urothelial carcinoma treated with neoadjuvant chemotherapy were noted to have a lower pathological stage distribution than patients treated with radical nephroureterectomy alone.

Subtyping Bladder Cancers: Biology vs Bioinformatics

Recent studies employing whole transcriptome expression data and unsupervised analytical methods concluded that bladder cancers can be grouped into basal and luminal molecular subtypes that have implications for prognostication and predicting response to therapy (1–7). Here Mo and colleagues used a supervised approach, based on their knowledge of biomarkers associated with normal differentiation, to assign bladder cancers from public data sets into two major molecular subtypes—basal and differentiated (8). Using this 18-gene biological classifier, they showed that the basal tumors were associated with shorter survival, consistent with their own previous observations (9) and those of the other groups (1–7). However, the relationship between basal subtype membership as defined by the 18-gene signature and poor clinical outcomes was stronger than was observed with some of the other classifiers (1–3), leading them to conclude that their classifier was a better tool for assigning bladder cancers to the basal subtype (“biology trumps pure bioinformatics”). While this may be true, it does not necessarily follow that the most accurate bladder cancer classifier will be the one that consistently shows that basal cancers are the most clinically aggressive. Recent studies concluded that patients with basal tumors obtained the most benefit from neoadjuvant chemotherapy (7,10), so the relative “aggressiveness” of basal tumors within a given cohort could be influenced by the relative proportion of chemosensitive basal tumors that are in it (and the MD Anderson “discovery” and The Cancer Genome Atlas (TCGA) cohorts contained some tumors from patients treated with neoadjuvant or adjuvant chemotherapy) (1,2). Immune checkpoint blockade will probably also complicate molecular subtype associations with survival (11). There is general consensus that, at the highest level, MIBCs can be segregated into basal and differentiated/luminal subtypes (12). However, there is also strong evidence that basal and luminal cancers can be subdivided further, and these subdivisions also appear to have clinical significance. For example, basal tumors can be segregated into “squamous” (TCGA cluster III) (1,6) and “claudin-low” (TCGA cluster IV) (3,7) subsets based on differential expression of biomarkers associated with epithelial-to-mesenchymal transition (EMT) (1,3,13), and the claudin-low tumors have unique T cell exhaustion signatures (13) and appear to be relatively resistant to neoadjuvant chemotherapy (7). Furthermore, the second TCGA article identifies another aggressive basal subset (“neuronal”) that may require unique therapeutic approaches (4). Similarly, the Lund group’s molecular taxonomy subdivides luminal tumors into “genomically unstable,” “infiltrated,” and “urothelial A” subsets (6,14), and the new TCGA marker article divides them into three clusters (4). A four-subtype classifier is now clinically available (7), so the relative value of separating basal and differentiated/ luminal bladder cancers into additional subsets will be apparent soon. The current study (8) also prompts consideration of whether an even simpler approach could be developed to distinguish basal from differentiated cancers. In breast cancer, immunohistochemistry with only three biomarkers (ER, PR, and HER2) largely captures the intrinsic subtypes defined by gene expression profiling (15), and conventional immunohistochemistry is still much easier to integrate into routine clinical practice than RNA expression-based platforms. This group (9) and teams at Lund University (16) and MD Anderson (17) have developed candidate immunohistochemical approaches for subtyping bladder cancers, and additional efforts are underway elsewhere. It would be interesting to know how much concordance would have been observed between the calls made by the 18-gene RNA classifier as compared with immunohistochemistry with antibodies specific to one basal biomarker (ie, KRT14) and one differentiated (ie, UPKs, FOXA1, GATA3, or PPARG) biomarker. The Baylor group’s own results suggest that a similar relationship to prognosis would be obtained (9). One of the most important unmet needs is to identify potentially lethal nonmuscle invasive bladder cancers (NMIBCs) so that they can be managed aggressively before they become a threat to the life of the patient. Various groups have identified biomarkers that may address this need, including a 12-gene panel that was recently validated in a large, prospective, multicenter study (18). One of the most interesting findings in the Mo

Ex vivo culture of tumor cells from N-methyl-N-nitrosourea-induced bladder cancer in rats: Development of organoids and an immortalized cell line

OBJECTIVE We ex vivo cultured primary tumor cells from N-methyl-N-nitrosourea (MNU)-induced bladder tumors in rats and established an immortalized cell line from them. MATERIALS AND METHODS Bladder tumors in rats were induced by instillation of MNU into the murine bladder. Primary tumor cells were prepared by the cancer-tissue originated spheroid method. An immortalized cell line was established by co-culture with fibroblasts. The cultured tumor cells were molecularly and functionally characterized by quantitative real-time polymerase chain reaction, Western blot, growth assay, and transwell migration assay. RESULTS Primary tumor cells were successfully prepared as multicellular spheroids from MNU-induced bladder tumors. The differentiation marker expression patterns observed in the original tumors were largely retained in the spheroids. We succeeded in establishing a cell line from the spheroids and named it T-MNU-1. Although basal markers (CK14 and CK5) were enriched in T-MNU-1 compared to the spheroids, T-MNU-1 expressed both luminal and basal markers. T-MNU-1 was able to migrate through a transwell. CONCLUSIONS Tumor cells in MNU-induced bladder tumors were successfully cultured ex vivo as organoids, and an immortalized cell line was also established from them. The ex vivo models offer a platform that enables analysis of intrinsic characteristics of tumor cells excluding influence of microenvironment in MNU-induced bladder tumors.

Dysregulation of EMT Drives the Progression to Clinically Aggressive Sarcomatoid Bladder Cancer

The sarcomatoid variant of urothelial bladder cancer (SARC) displays a high propensity for distant metastasis and is associated with short survival. We report a comprehensive genomic analysis of 28 cases of SARCs and 84 cases of conventional urothelial carcinomas (UCs), with the TCGA cohort of 408 muscle-invasive bladder cancers serving as the reference. SARCs showed a distinct mutational landscape with enrichment of TP53, RB1, and PIK3CA mutations. They were related to the basal molecular subtype of conventional UCs and could be divided into epithelial/basal and more clinically aggressive mesenchymal subsets based on TP63 and its target genes expression levels. Other analyses revealed that SARCs are driven by downregulation of homotypic adherence genes and dysregulation of cell cycle and EMT networks, and nearly half exhibited a heavily infiltrated immune phenotype. Our observations have important implications for prognostication and the development of more effective therapies for this highly lethal variant of bladder cancer.