The clonal relation of primary upper urinary tract urothelial carcinoma and paired urothelial carcinoma of the bladder

The risk of developing urothelial carcinoma of the bladder (UCB) in patients treated by radical nephroureterectomy (RNU) for an upper urinary tract urothelial carcinoma (UTUC) is 22% to 47% in the 2 years after surgery. Subject of debate remains whether UTUC and the subsequent UCB are clonally related or represent separate origins. To investigate the clonal relationship between both entities, we performed targeted DNA sequencing of a panel of 41 genes on matched normal and tumor tissue of 15 primary UTUC patients treated by RNU who later developed 19 UCBs. Based on the detected tumor-specific DNA aberrations, the paired UTUC and UCB(s) of 11 patients (73.3%) showed a clonal relation, whereas in four patients the molecular results did not indicate a clear clonal relationship. Our results support the hypothesis that UCBs following a primary surgically resected UTUC are predominantly clonally derived recurrences and not separate entities

Genome-wide aneuploidy detected by mFast-SeqS in circulating cell-free DNA is associated with poor response to pembrolizumab in patients with advanced urothelial cancer

Second-line treatment with immune checkpoint inhibition in patients with metastatic urothelial cancer (mUC) has a low success rate (~ 20%). Circulating tumour-derived DNA (ctDNA) levels may guide patient stratification, provided that an affordable and robust assay is available. Here, we investigate whether the modified fast aneuploidy screening test-sequencing system (mFast-SeqS) may provide such an assay. To this end, mFast-SeqS was performed on cell-free DNA (cfDNA) from 74 patients with mUC prior to treatment with pembrolizumab. Results were associated with corresponding tissue-based profiles, plasma-based variant allele frequencies (VAFs) and clinical response. We found that plasma-derived mFast-SeqS-based aneuploidy scores significantly correlated with those observed in the corresponding tumour tissue as well as with the ctDNA level in the plasma. In multivariate logistic regression analysis, a high aneuploidy score was independently associated with lack of clinical benefit from treatment with pembrolizumab. In conclusion, mFast-SeqS provides a patient-friendly, high-throughput and affordable method to estimate ctDNA level. Following independent validation, this test could be used to stratify mUC patients for response prior to the initiation of treatment with pembrolizumab

Genome-wide aneuploidy detected by mFast-SeqS in circulating cell-free DNA is associated with poor response to pembrolizumab in patients with advanced urothelial cancer

Second‐line treatment with immune checkpoint inhibition in patients with metastatic urothelial cancer (mUC) has a low success rate (~ 20%). Circulating tumour‐derived DNA (ctDNA) levels may guide patient stratification, provided that an affordable and robust assay is available. Here, we investigate whether the modified fast aneuploidy screening test‐sequencing system (mFast‐SeqS) may provide such an assay. To this end, mFast‐SeqS was performed on cell‐free DNA (cfDNA) from 74 patients with mUC prior to treatment with pembrolizumab. Results were associated with corresponding tissue‐based profiles, plasma‐based variant allele frequencies (VAFs) and clinical response. We found that plasma‐derived mFast‐SeqS‐based aneuploidy scores significantly correlated with those observed in the corresponding tumour tissue as well as with the ctDNA level in the plasma. In multivariate logistic regression analysis, a high aneuploidy score was independently associated with lack of clinical benefit from treatment with pembrolizumab. In conclusion, mFast‐SeqS provides a patient‐friendly, high‐throughput and affordable method to estimate ctDNA level. Following independent validation, this test could be used to stratify mUC patients for response prior to the initiation of treatment with pembrolizumab

Anti-PD-1 Efficacy in Patients with Metastatic Urothelial Cancer Associates with Intratumoral Juxtaposition of T Helper-Type 1 and CD8(+) T cells

Purpose: PD-1 inhibition results in durable antitumor responses in a proportion of patients with metastatic urothelial cancer (mUC). The majority of patients, however, do not experience clinical benefit. In this study, we aimed to identify early changes in T-cell subsets that underlie anti-PD-1 efficacy in patients with mUC.Experimental Design: Paired samples were collected from peripheral blood, plasma, and metastatic lesions of 56 patients with mUC at baseline and weeks 6 and 12 after initiating pembrolizumab treatment (200 mg intravenously, every 3 weeks). Samples were analyzed using multiplex flow cytometry, ELISA, and in situ stainings, including cellular network analysis. Treatment response was evaluated as best overall response according to RECIST v1.1, and patients were classified as responder (complete or partial response) or nonresponder (progressive disease).Results: In responders, baseline fractions of CD4(+) T cells expressing cosignaling receptors were higher compared with nonresponders. The fraction of circulating PD-1(+) CD4(+) T cells decreased at weeks 6 and 12, whereas the fraction of 4-1BB(+) CD28(+) CD4(+) T cells increased at week 12. In metastatic lesions of responders, the baseline density of T helper-type 1 (Th1) cells, defined as T-bet(+) CD4(+) T cells, was higher as compared to non-responders. Upon treatment, Th1 cells became localized in close proximity to CD8(+) T cells, CD11b(+) myeloid cells, and tumor cells.Conclusions: A decrease in the fraction of circulating PD-1(+) CD4(+) T cells, and juxtaposition of Th1, CD8(+), and myeloid cells was associated with response to anti-PD-1 treatment in patients with mUC

Anti–PD-1 Efficacy in Patients with Metastatic Urothelial Cancer Associates with Intratumoral Juxtaposition of T Helper-Type 1 and CD8 þ T cells

Purpose: PD-1 inhibition results in durable antitumor responses in a proportion of patients with metastatic urothelial cancer (mUC). The majority of patients, however, do not experience clinical benefit. In this study, we aimed to identify early changes in T-cell subsets that underlie anti–PD-1 efficacy in patients with mUC. Experimental Design: Paired samples were collected from peripheral blood, plasma, and metastatic lesions of 56 patients with mUC at baseline and weeks 6 and 12 after initiating pembrolizumab treatment (200 mg intravenously, every 3 weeks). Samples were analyzed using multiplex flow cytometry, ELISA, and in situ stainings, including cellular network analysis. Treatment response was evaluated as best overall response according to RECIST v1.1, and patients were classified as responder (complete or partial response) or nonresponder (progressive disease). Results: In responders, baseline fractions of CD4 þ T cells expressing cosignaling receptors were higher compared with nonresponders. The fraction of circulating PD-1 þ CD4 þ T cells decreased at weeks 6 and 12, whereas the fraction of 4-1BB þ CD28 þ CD4 þ T cells increased at week 12. In metastatic lesions of responders, the baseline density of T helper-type 1 (Th1) cells, defined as T-bet þ CD4 þ T cells, was higher as compared to nonresponders. Upon treatment, Th1 cells became localized in close proximity to CD8 þ T cells, CD11b þ myeloid cells, and tumor cells. Conclusions: A decrease in the fraction of circulating PD-1 þ CD4 þ T cells, and juxtaposition of Th1, CD8 þ, and myeloid cells was associated with response to anti–PD-1 treatment in patients with mUC

Anti–PD-1 Efficacy in Patients with Metastatic Urothelial Cancer Associates with Intratumoral Juxtaposition of T Helper-Type 1 and CD8þ T cells

Purpose: PD-1 inhibition results in durable antitumor responses in a proportion of patients with metastatic urothelial cancer (mUC). The majority of patients, however, do not experience clinical benefit. In this study, we aimed to identify early changes in T-cell subsets that underlie anti–PD-1 efficacy in patients with mUC. Experimental Design: Paired samples were collected from peripheral blood, plasma, and metastatic lesions of 56 patients with mUC at baseline and weeks 6 and 12 after initiating pembrolizumab treatment (200 mg intravenously, every 3 weeks). Samples were analyzed using multiplex flow cytometry, ELISA, and in situ stainings, including cellular network analysis. Treatment response was evaluated as best overall response according to RECIST v1.1, and patients were classified as responder (complete or partial response) or nonresponder (progressive disease). Results: In responders, baseline fractions of CD4þ T cells expressing cosignaling receptors were higher compared with nonresponders. The fraction of circulating PD-1þ CD4þ T cells decreased at weeks 6 and 12, whereas the fraction of 4-1BBþ CD28þ CD4þ T cells increased at week 12. In metastatic lesions of responders, the baseline density of T helper-type 1 (Th1) cells, defined as T-betþ CD4þ T cells, was higher as compared to nonresponders. Upon treatment, Th1 cells became localized in close proximity to CD8þ T cells, CD11bþ myeloid cells, and tumor cells. Conclusions: A decrease in the fraction of circulating PD-1þ CD4þ T cells, and juxtaposition of Th1, CD8þ, and myeloid cells was associated with response to anti–PD-1 treatment in patients with mUC

Gene-expression-based T-Cell-to-Stroma Enrichment (TSE) score predicts response to immune checkpoint inhibitors in urothelial cancer

Abstract Immune checkpoint inhibitors (ICI) improve overall survival in patients with metastatic urothelial cancer (mUC), but therapeutic success at the individual patient level varies significantly. Here we identify predictive markers of response, based on whole-genome DNA (n = 70) and RNA-sequencing (n = 41) of fresh metastatic biopsy samples, collected prior to treatment with pembrolizumab. We find that PD-L1 combined positivity score does not, whereas tumor mutational burden and APOBEC mutagenesis modestly predict response. In contrast, T cell-to-stroma enrichment (TSE) score, computed from gene expression signature data to capture the relative abundance of T cells and stromal cells, predicts response to immunotherapy with high accuracy. Patients with a positive and negative TSE score show progression free survival rates at 6 months of 67 and 0%, respectively. The abundance of T cells and stromal cells, as reflected by the TSE score is confirmed by immunofluorescence in tumor tissue, and its good performance in two independent ICI-treated cohorts of patients with mUC (IMvigor210) and muscle-invasive UC (ABACUS) validate the predictive power of the TSE score. In conclusion, the TSE score represents a clinically applicable metric that potentially supports the prospective selection of patients with mUC for ICI treatment