Early On-treatment Circulating Tumor DNA Measurements and Response to Immune Checkpoint Inhibitors in Advanced Urothelial Cancer.

BACKGROUND: Immune checkpoint inhibitors (ICIs) can induce durable disease control in metastatic urothelial cancer (mUC), but only 20-25% of patients respond. Early identification of a nondurable response will improve management strategies. OBJECTIVE: To investigate whether on-treatment circulating tumor DNA (ctDNA) measurements can predict ICI responsiveness in mUC patients. DESIGN, SETTING, AND PARTICIPANTS: This study consists of a discovery cohort of 40 mUC patients and a prospective multicenter validation cohort of 16 mUC patients. Plasma cell-free DNA was collected at baseline and after 3 and 6 wk on ICIs. The ctDNA levels were calculated from targeted sequencing. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Outcome measurements were progression-free survival (PFS), overall survival (OS), and nondurable response (PFS ≤6 mo). Relationships with ctDNA were assessed using Cox regression. Changes in ctDNA level at 3 and 6 wk were categorized by an increase or decrease relative to baseline. RESULTS AND LIMITATIONS: In the discovery cohort, ctDNA was detected in 37/40 (93%) of patients at baseline. A ctDNA increase was observed in 12/15 (80%) and ten of 12 (83%) patients with a nondurable response at 3 and 6 wk, respectively. Of patients with a durable response (PFS >6 mo), 94% showed a decrease. A ctDNA increase at 3 wk was associated with shorter PFS (hazard ratio [HR] 7.8, 95% confidence interval [CI] 3.1-19.5) and OS (HR 8.0, 95% CI 3.0-21.0), independent of clinical prognostic variables. Similar results were observed at 6 wk. The 3-wk association with PFS was validated in a prospective cohort (HR 7.5, 95% CI 1.3-42.6). Limitations include the limited number of patients. CONCLUSIONS: Early changes in ctDNA levels are strongly linked to the duration of ICI benefit in mUC and may contribute to timely therapy modifications. PATIENT SUMMARY: Benefit from immunotherapy can be predicted after only 3 wk of treatment by investigating cancer DNA in blood. This could help in timely therapy changes for urothelial cancer patients with limited benefit from immunotherapy.

Intratumoral T cell depletion following neoadjuvant chemotherapy in patients with muscle-invasive bladder cancer is associated with poor clinical outcome.

BACKGROUND: Whereas neoadjuvant cisplatin-based chemotherapy (NAC) followed by a radical cystectomy remains the standard treatment for patients with muscle-invasive bladder cancer (MIBC), increasing evidence suggests that checkpoint inhibitors, either alone or in combination with chemotherapy, are effective in the (neo)adjuvant setting. The major aim of this study was to improve our understanding of the immune-modulating effects of NAC in MIBC. METHODS: Tumor tissue of 81 patients was used, including 60 patients treated with NAC and 21 patients undergoing upfront cystectomy. Multiplex immunohistochemistry was performed to assess CD3+, CD3+CD8+, CD3+CD8-FoxP3-, CD3+FoxP3+, and CD20+ cells. Patients were classified into a favorable or unfavorable outcome group based on the development of a recurrence within a year. RESULTS: The density of intratumoral CD3+ T cells decreased following NAC in patients with a recurrence at one year, while it remained stable in patients without a recurrence (median fold change 0.6 [95CI 0.3; 1.0] versus 1.0 [95CI 0.6; 2.2]). This decrease was mainly attributable to a decrease in CD3+CD8-FoxP3- and CD3+FoxP3+ T cells and was not observed in patients with an early recurrence after upfront cystectomy. Additionally, in cystectomy tissue of patients treated with NAC, median CD3+ and CD3+CD8+ T cell densities were significantly lower in patients with versus patients without a recurrence (CD3: 261. cells/mm2 [95CI 22.4; 467.2]; CD8: 189.6 cells/mm2 [95CI 2.0;462.0]). CONCLUSION: T cell density decreases following NAC in MIBC patients with poor clinical outcome. Further research is needed to investigate whether this decrease in T cell density affects the efficacy of subsequent checkpoint inhibitors. PRéCIS: The major aim of this study was to improve our understanding of the immune-modulating effects of NAC in patients with MIBC. We reveal a decline in intratumoral CD3+ T cell density following NAC in patients with an early recurrence.

Detection and localization of early- and late-stage cancers using platelet RNA.

Cancer patients benefit from early tumor detection since treatment outcomes are more favorable for less advanced cancers. Platelets are involved in cancer progression and are considered a promising biosource for cancer detection, as they alter their RNA content upon local and systemic cues. We show that tumor-educated platelet (TEP) RNA-based blood tests enable the detection of 18 cancer types. With 99% specificity in asymptomatic controls, thromboSeq correctly detected the presence of cancer in two-thirds of 1,096 blood samples from stage I-IV cancer patients and in half of 352 stage I-III tumors. Symptomatic controls, including inflammatory and cardiovascular diseases, and benign tumors had increased false-positive test results with an average specificity of 78%. Moreover, thromboSeq determined the tumor site of origin in five different tumor types correctly in over 80% of the cancer patients. These results highlight the potential properties of TEP-derived RNA panels to supplement current approaches for blood-based cancer screening.

Homologous recombination repair deficient prostate cancer represents an immunologically distinct subtype.

Homologous recombination repair deficiency (HRD) is observed in 10% of patients with castrate-resistant prostate cancer (PCa). Preliminary data suggest that HRD-PCa might be more responsive to immune checkpoint inhibitors (ICIs). In this study, we compare the tumor immune landscape and peripheral T cell receptor (TCR) repertoire of patients with and without HRD-PCa to gain further insight into the immunogenicity of HRD-PCa. Immunohistochemistry was performed on tumor tissue of 81 patients, including 15 patients with HRD-PCa. Peripheral TCR sequencing was performed in a partially overlapping cohort of 48 patients, including 16 patients with HRD-PCa. HRD patients more frequently had intratumoral CD3+, CD3+CD8-FoxP3- or Foxp3+ TILs above median compared to patients without DNA damage repair alterations (DDRwt; CD3+ and Foxp3+: 77% vs 35%, p = .013; CD3+CD8-FoxP3-: 80% vs 44%, p = .031). No significant difference in CD8+ TILs or PD-L1 expression was observed. In peripheral blood, HRD patients displayed a more diverse TCR repertoire compared to DDRwt patients (p = .014). Additionally, HRD patients shared TCR clusters with low generation probability, suggesting patient-overlapping T cell responses. A pooled analysis of clinical data from 227 patients with molecularly characterized PCa suggested increased efficacy of ICIs in HRD-PCa. In conclusion, patients with HRD-PCa display increased TIL density and an altered peripheral TCR repertoire. Further research into the efficacy of ICIs and the presence of shared neoantigens in HRD-PCa is warranted.

Comprehensive Molecular Characterization Reveals Genomic and Transcriptomic Subtypes of Metastatic Urothelial Carcinoma.

Recent molecular characterization of primary urothelial carcinoma (UC) may guide future clinical decision-making. For metastatic UC (mUC), a comprehensive molecular characterization is still lacking. We analyzed whole-genome DNA and RNA sequencing data for fresh-frozen metastatic tumor biopsies from 116 mUC patients who were scheduled for palliative systemic treatment within the context of a clinical trial (NCT01855477 and NCT02925234). Hierarchical clustering for mutational signatures revealed two major genomic subtypes: GenS1 (67%), which was APOBEC-driven; and GenS2 (24%), which had a high fraction of de novo mutational signatures related to reactive oxygen species and is putatively clock-like. Significantly mutated genes (SMGs) did not differ between the genomic subtypes. Transcriptomic analysis revealed five mUC subtypes: luminal-a and luminal-b (40%), stroma-rich (24%), basal/squamous (23%), and a nonspecified subtype (12%). These subtypes differed regarding expression of key genes, SMGs, oncogenic pathway activity, and immune cell infiltration. We integrated the genomic and transcriptomic data to propose potential therapeutic options by transcriptomic subtype and for individual patients. This in-depth analysis of a large cohort of patients with mUC may serve as a reference for subtype-oriented and patient-specific research on the etiology of mUC and for novel drug development. PATIENT SUMMARY: We carried out an in-depth analysis of the molecular and genetic features of metastatic cancer involving the cells that line the urinary tract. We showed that this is a heterogeneous disease with different molecular subtypes and we identified possible targets for therapy for each subtype.

Whole Blood Transcriptome Profiling Identifies DNA Replication and Cell Cycle Regulation as Early Marker of Response to Anti-PD-1 in Patients with Urothelial Cancer.

Although immune checkpoint inhibitors improve median overall survival in patients with metastatic urothelial cancer (mUC), only a minority of patients benefit from it. Early blood-based response biomarkers may provide a reliable way to assess response weeks before imaging is available, enabling an early switch to other therapies. We conducted an exploratory study aimed at the identification of early markers of response to anti-PD-1 in patients with mUC. Whole blood RNA sequencing and phenotyping of peripheral blood mononuclear cells were performed on samples of 26 patients obtained before and after 2 to 6 weeks of anti-PD-1. Between baseline and on-treatment samples of patients with clinical benefit, 51 differentially expressed genes (DEGs) were identified, of which 37 were upregulated during treatment. Among the upregulated genes was PDCD1, the gene encoding PD-1. STRING network analysis revealed a cluster of five interconnected DEGs which were all involved in DNA replication or cell cycle regulation. We hypothesized that the upregulation of DNA replication/cell cycle genes is a result of T cell proliferation and we were able to detect an increase in Ki-67+ CD8+ T cells in patients with clinical benefit (median increase: 1.65%, range -0.63 to 7.06%, p = 0.012). In patients without clinical benefit, no DEGs were identified and no increase in Ki-67+ CD8+ T cells was observed. In conclusion, whole blood transcriptome profiling identified early changes in DNA replication and cell cycle regulation genes as markers of clinical benefit to anti-PD-1 in patients with urothelial cancer. Although promising, our findings require further validation before implementation in the clinic.

Immunophenotyping Reveals Longitudinal Changes in Circulating Immune Cells During Radium-223 Therapy in Patients With Metastatic Castration-Resistant Prostate Cancer.

BACKGROUND: Radium-223 improves overall survival (OS) in men with bone metastatic castration-resistant prostate cancer (mCRPC). While the exact mechanism behind this survival benefit remains unclear, radium-induced immunological mechanisms might contribute to the OS advantage. We performed a comprehensive evaluation of the immunological changes in mCRPC patients by phenotyping the peripheral blood mononuclear cells (PBMCs) during radium-223 therapy. MATERIALS AND METHODS: In this prospective, single-arm, exploratory study, PBMCs of 30 mCRPC patients were collected before, during, and after treatment with radium-223. Lymphocyte and monocyte counts were analyzed to get insight into general immune cell trends. Next, we analyzed changes in T cell subsets, myeloid-derived suppressor cells (MDSCs), and immune checkpoint expression using linear regression models. Per subset, the 6-month change (% of baseline) was determined. Bootstrapped 95% confidence intervals were used to measure the degree of uncertainty of our findings. RESULTS: We observed a substantial decrease in absolute lymphocyte counts (-0.12 * 10^9 cells/L per injection, 95% CI: -0.143 - -0.102). Simultaneously, an increase was observed in the proportion of T cells that expressed costimulatory (ICOS) or inhibitory (TIM-3, PD-L1, and PD-1) checkpoint molecules. Moreover, the fraction of two immunosuppressive subsets - the regulatory T cells and the monocytic MDSCs - increased throughout treatment. These findings were not more pronounced in patients with an alkaline phosphatase response during therapy. CONCLUSION: Immune cell subsets in patients with mCRPC changed during radium-223 therapy, which warrants further research into the possible immunological consequences of these changes.

Homologous Recombination Repair Deficiency and Implications for Tumor Immunogenicity.

Homologous recombination repair deficiency (HRD) can be observed in virtually all cancer types. Although HRD sensitizes tumors to DNA-damaging chemotherapy and poly(ADP-ribose) polymerase (PARP) inhibitors, all patients ultimately develop resistance to these therapies. Therefore, it is necessary to identify therapeutic regimens with a more durable efficacy. HRD tumors have been suggested to be more immunogenic and, therefore, more susceptible to treatment with checkpoint inhibitors. In this review, we describe how HRD might mechanistically affect antitumor immunity and summarize the available translational evidence for an association between HRD and antitumor immunity across multiple tumor types. In addition, we give an overview of all available clinical data on the efficacy of checkpoint inhibitors in HRD tumors and describe the evidence for using treatment strategies that combine checkpoint inhibitors with PARP inhibitors.

Spatial and Temporal Heterogeneity of Tumor-Infiltrating Lymphocytes in Advanced Urothelial Cancer.

Checkpoint inhibitors targeting PD-(L)1 induce objective responses in 20% of patients with metastatic urothelial cancer (UC). CD8+ T cell infiltration has been proposed as a putative biomarker for response to checkpoint inhibitors. Nevertheless, data on spatial and temporal heterogeneity of tumor-infiltrating lymphocytes in advanced UC are lacking. The major aims of this study were to explore spatial heterogeneity for lymphocyte infiltration and to investigate how the immune landscape changes during the disease course. We performed multiplex immunohistochemistry to assess the density of intratumoral and stromal CD3+, CD8+, FoxP3+ and CD20+ immune cells in longitudinally collected samples of 49 UC patients. Within these samples, spatial heterogeneity for lymphocyte infiltration was observed. Regions the size of a 0.6 tissue microarray core (0.28 mm2) provided a representative sample in 60.6 to 71.6% of cases, depending on the cell type of interest. Regions of 3.30 mm2, the median tumor surface area in our biopsies, were representative in 58.8 to 73.8% of cases. Immune cell densities did not significantly differ between untreated primary tumors and metachronous distant metastases. Interestingly, CD3+, CD8+ and FoxP3+ T cell densities decreased during chemotherapy in two small cohorts of patients treated with neoadjuvant or palliative platinum-based chemotherapy. In conclusion, spatial heterogeneity in advanced UC challenges the use of immune cell infiltration in biopsies as biomarker for response prediction. Our data also suggests a decrease in tumor-infiltrating T cells during platinum-based chemotherapy.

Prognostic and Predictive Value of Tumor-Infiltrating Immune Cells in Urothelial Cancer of the Bladder.

The prognosis and responsiveness to chemotherapy and checkpoint inhibitors differs substantially among patients with bladder cancer (BC). There is an unmet need for biomarkers that can accurately predict prognosis and treatment outcome. Here, we describe the available literature on the prognostic and predictive value of tumor-infiltrating immune cells in BC. Current evidence indicates that a high density of tumor-infiltrating CD8+ T cells is a favorable prognostic factor, whereas PD-L1 expression and tumor-associated macrophages are unfavorable prognostic features. While PD-L1 expression appears unsuccessful as a biomarker for the response to checkpoint inhibitors, there are some indications that high CD8+ T cell infiltration, low transforming growth factor-beta signaling and low densities of myeloid-derived suppressor cells are associated with response. Future studies should focus on combinations of biomarkers to accurately predict survival and response to treatment.

Lactate dehydrogenase: a marker of diminished antitumor immunity.

Lactate dehydrogenase (LDH) levels are inversely related with response to checkpoint inhibitors. Elevated LDH levels are the product of enhanced glycolytic activity of the tumor and tumor necrosis due to hypoxia, the latter being associated with high tumor burden. In this review, we elucidate the effects of glycolysis and hypoxia on antitumor immunity and set forth ways to improve response to immunotherapy in cancer patients with elevated LDH levels. We discuss the current knowledge on combining immunotherapy with glycolysis inhibitors, anti-acidifying drugs, anti-angiogenic or cytoreductive therapy.

Immune checkpoint inhibition-related colitis: symptoms, endoscopic features, histology and response to management.

BACKGROUND: Immune checkpoint inhibitors are successfully introduced as anticancer treatment. However, they may induce severe immune-related adverse events (irAEs). One of the most frequent irAEs is diarrhoea. The main objective of this study was to analyse symptoms (ie, grade of diarrhoea), endoscopic and histological features and response to management in immune checkpoint inhibition-related colitis (IRC). PATIENTS AND METHODS: We retrospectively analysed patients who developed diarrhoea on checkpoint inhibition and therefore underwent an endoscopy and/or were treated with corticosteroids. Patients were treated between August 2010 and March 2016 for metastatic melanoma or non-small cell lung cancer. Severity of IRC was scored using the endoscopic Mayo score and the van der Heide score. RESULTS: Out of a cohort of 781 patients, 92 patients were identified who developed diarrhoea and therefore underwent an endoscopy and/or were treated with corticosteroids. Patients were treated with monotherapy anticytotoxic T-lymphocyte antigen-4, antiprogrammed death receptor-1 or a combination of both. All patients had symptoms of diarrhoea (grade 1: 16%; grade 2: 39% and grade 3: 44%). A complete colonoscopy was performed in 62 (67%) patients, of whom 42 (68%) had a pancolitis (≥3 affected segments). Ulcers were seen in 32% of endoscopies. There was no significant correlation between the grade of diarrhoea at presentation and endoscopic severity scores, the presence of ulcers or histological features. In 54 episodes of diarrhoea (56%), patients received one or more cycles infliximab for steroid-refractory colitis. Patients with higher endoscopic severity scores, ulcers and/or a pancolitis needed infliximab more often. CONCLUSIONS: The correlation between grade of diarrhoea and endoscopic or histological features for severity of colitis is poor. Patients with higher endoscopic severity scores, ulcers or a pancolitis needed the addition of infliximab more often. Therefore, endoscopy may have value in the evaluation of the severity of IRC and may help in decision making for optimal management.

Impact of disease location on fecal calprotectin levels in Crohn's disease.

OBJECTIVE: The correlation between the Simple Endoscopic Score for Crohn's Disease (SES-CD) and fecal calprotectin is well established in (ileo)colonic Crohn's disease (CD). However, for ileal CD, existing data are conflicting. The aim of this study is to evaluate the biomarker profile in CD patients with varying severity and location of mucosal ulceration. MATERIALS AND METHODS: An electronic patient database search identified CD patients in whom ileocolonoscopy, fecal calprotectin (CALPRO), serum C-reactive protein (CRP) and blood leukocyte counts (LEU) were measured within a 4-week interval without changes in medication. Ileocolonoscopies were scored for the presence of ulcers in each segment as defined by the SES-CD and the sum of segmental ulcer scores resulted in a partial SES-CD (pSES-CD). RESULTS: Fourty-four patients were identified, of whom 9/44 had ileal CD, 20/44 colonic and 15/44 ileocolonic CD based on the Montreal classification. In the total study population CALPRO correlated best with pSES-CD (r = 0.76, p < 0.0001), followed by LEU (r = 0.54, p = 0.0004) and CRP (r = 0.45, p = 0.0026). Patients with ileal CD had significantly lower CALPRO level than those with (ileo)colonic disease even in the presence of large and/or very large ulcers (mean ± SEM: 297 ± 81 µg/g vs. 1523 ± 97 µg/g, p < 0.0001). LEU was also significantly lower in the presence of large and/or very large ulcers in ileal CD compared to those with (ileo)colonic disease (mean ± SEM: 6.7 ± 0.9 × 10(9)/l vs. 10.6 ± 0.8 × 10(9)/l, p = 0.02). A similar trend was identified regarding CRP levels. CONCLUSIONS: Even in the presence of large or very large ulcers, patients with ileal Crohn's may not have markedly elevated fecal calprotectin levels.

Specificity of binding of the plectin actin-binding domain to beta4 integrin.

Plectin is a major component of the cytoskeleton and links the intermediate filament system to hemidesmosomes by binding to the integrin beta4 subunit. Previously, a binding site for beta4 was mapped on the actin-binding domain (ABD) of plectin and binding of beta4 and F-actin to plectin was shown to be mutually exclusive. Here we show that only the ABDs of plectin and dystonin bind to beta4, whereas those of other actin-binding proteins do not. Mutations of the ABD of plectin-1C show that Q131, R138, and N149 are critical for tight binding of the ABD to beta4. These residues form a small cavity, occupied by a well-ordered water molecule in the crystal structure. The beta4 binding pocket partly overlaps with the actin-binding sequence 2 (ABS2), previously shown to be essential for actin binding. Therefore, steric interference may render binding of beta4 and F-actin to plectin mutually exclusive. Finally, we provide evidence indicating that the residues preceding the ABD in plectin-1A and -1C, although unable to mediate binding to beta4 themselves, modulate the binding activity of the ABD for beta4. These studies demonstrate the unique property of the plectin-ABD to bind to both F-actin and beta4, and explain why several other ABD-containing proteins that are expressed in basal keratinocytes are not recruited into hemidesmosomes.