Integrative clinical genomics of advanced prostate cancer.

Toward development of a precision medicine framework for metastatic, castration-resistant prostate cancer (mCRPC), we established a multi-institutional clinical sequencing infrastructure to conduct prospective whole-exome and transcriptome sequencing of bone or soft tissue tumor biopsies from a cohort of 150 mCRPC affected individuals. Aberrations of AR, ETS genes, TP53, and PTEN were frequent (40%-60% of cases), with TP53 and AR alterations enriched in mCRPC compared to primary prostate cancer. We identified new genomic alterations in PIK3CA/B, R-spondin, BRAF/RAF1, APC, ß-catenin, and ZBTB16/PLZF. Moreover, aberrations of BRCA2, BRCA1, and ATM were observed at substantially higher frequencies (19.3% overall) compared to those in primary prostate cancers. 89% of affected individuals harbored a clinically actionable aberration, including 62.7% with aberrations in AR, 65% in other cancer-related genes, and 8% with actionable pathogenic germline alterations. This cohort study provides clinically actionable information that could impact treatment decisions for these affected individuals.

Integrated single-cell genetic and transcriptional analysis suggests novel drivers of chronic lymphocytic leukemia.

Intra-tumoral genetic heterogeneity has been characterized across cancers by genome sequencing of bulk tumors, including chronic lymphocytic leukemia (CLL). In order to more accurately identify subclones, define phylogenetic relationships, and probe genotype-phenotype relationships, we developed methods for targeted mutation detection in DNA and RNA isolated from thousands of single cells from five CLL samples. By clearly resolving phylogenic relationships, we uncovered mutated LCP1 and WNK1 as novel CLL drivers, supported by functional evidence demonstrating their impact on CLL pathways. Integrative analysis of somatic mutations with transcriptional states prompts the idea that convergent evolution generates phenotypically similar cells in distinct genetic branches, thus creating a cohesive expression profile in each CLL sample despite the presence of genetic heterogeneity. Our study highlights the potential for single-cell RNA-based targeted analysis to sensitively determine transcriptional and mutational profiles of individual cancer cells, leading to increased understanding of driving events in malignancy.

Cell cycle-targeting microRNAs promote differentiation by enforcing cell-cycle exit.

MicroRNAs (miRNAs) have been known to affect various biological processes by repressing expression of specific genes. Here we describe an essential function of the miR-34/449 family during differentiation of epithelial cells. We found that miR-34/449 suppresses the cell-cycle machinery in vivo and promotes cell-cycle exit, thereby allowing epithelial cell differentiation. Constitutive ablation of all six members of this miRNA family causes derepression of multiple cell cycle-promoting proteins, thereby preventing epithelial cells from exiting the cell cycle and entering a quiescent state. As a result, formation of motile multicilia is strongly inhibited in several tissues such as the respiratory epithelium and the fallopian tube. Consequently, mice lacking miR-34/449 display infertility as well as severe chronic airway disease leading to postnatal death. These results demonstrate that miRNA-mediated repression of the cell cycle is required to allow epithelial cell differentiation.

Embryonic transcription factor SOX9 drives breast cancer endocrine resistance.

The estrogen receptor (ER) drives the growth of most luminal breast cancers and is the primary target of endocrine therapy. Although ER blockade with drugs such as tamoxifen is very effective, a major clinical limitation is the development of endocrine resistance especially in the setting of metastatic disease. Preclinical and clinical observations suggest that even following the development of endocrine resistance, ER signaling continues to exert a pivotal role in tumor progression in the majority of cases. Through the analysis of the ER cistrome in tamoxifen-resistant breast cancer cells, we have uncovered a role for an RUNX2-ER complex that stimulates the transcription of a set of genes, including most notably the stem cell factor SOX9, that promote proliferation and a metastatic phenotype. We show that up-regulation of SOX9 is sufficient to cause relative endocrine resistance. The gain of SOX9 as an ER-regulated gene associated with tamoxifen resistance was validated in a unique set of clinical samples supporting the need for the development of improved ER antagonists.

Genome-wide CRISPR screen identifies HNRNPL as a prostate cancer dependency regulating RNA splicing.

Alternative RNA splicing plays an important role in cancer. To determine which factors involved in RNA processing are essential in prostate cancer, we performed a genome-wide CRISPR/Cas9 knockout screen to identify the genes that are required for prostate cancer growth. Functional annotation defined a set of essential spliceosome and RNA binding protein (RBP) genes, including most notably heterogeneous nuclear ribonucleoprotein L (HNRNPL). We defined the HNRNPL-bound RNA landscape by RNA immunoprecipitation coupled with next-generation sequencing and linked these RBP-RNA interactions to changes in RNA processing. HNRNPL directly regulates the alternative splicing of a set of RNAs, including those encoding the androgen receptor, the key lineage-specific prostate cancer oncogene. HNRNPL also regulates circular RNA formation via back splicing. Importantly, both HNRNPL and its RNA targets are aberrantly expressed in human prostate tumors, supporting their clinical relevance. Collectively, our data reveal HNRNPL and its RNA clients as players in prostate cancer growth and potential therapeutic targets.

Circulating inflammation markers and prostate cancer.

BACKGROUND: Chronic inflammation is thought to influence the risk of prostate cancer. The purpose of this population-based case-control study was to evaluate the association of 48 circulating inflammation markers with prostate cancer, to identify candidate markers for further investigation. METHODS: Serum samples collected from 235 prostate cancer patients and 198 population-based controls recruited in Örebro County, Sweden, in 1989-1991, were assessed using a multiplex bead-based immunoassay to determine concentrations of 48 circulating inflammation markers. Logistic regression was first used to evaluate the association between individual markers (highest vs lowest concentration quartile) and prostate cancer in unadjusted and mutually adjusted models. Second, patients with inflammatory conditions, metastatic or advanced prostate cancer, were excluded to address the possible influence of systemic disease on inflammation markers. RESULTS: Individual analyses first identified 21 markers associated with prostate cancer (P < .05), which after mutual adjustment were reduced to seven markers. After the exclusion of men with conditions linked with systemic inflammation, associations between prostate cancer and deviant levels of C-X3-C motif chemokine ligand 1, platelet-derived growth factor subunit B homodimer, interleukin 10, C-C motif chemokine ligand (CCL) 21, and CCL11 remained statistically significant. CONCLUSIONS: In this explorative study, we identified candidate inflammation markers of possible importance for prostate cancer pathophysiology, for further evaluation in prospective studies.

Ipilimumab for Patients with Relapse after Allogeneic Transplantation.

BACKGROUND: Loss of donor-mediated immune antitumor activity after allogeneic hematopoietic stem-cell transplantation (HSCT) permits relapse of hematologic cancers. We hypothesized that immune checkpoint blockade established by targeting cytotoxic T-lymphocyte-associated protein 4 with ipilimumab could restore antitumor reactivity through a graft-versus-tumor effect. METHODS: We conducted a phase 1/1b multicenter, investigator-initiated study to determine the safety and efficacy of ipilimumab in patients with relapsed hematologic cancer after allogeneic HSCT. Patients received induction therapy with ipilimumab at a dose of 3 or 10 mg per kilogram of body weight every 3 weeks for a total of 4 doses, with additional doses every 12 weeks for up to 60 weeks in patients who had a clinical benefit. RESULTS: A total of 28 patients were enrolled. Immune-related adverse events, including one death, were observed in 6 patients (21%), and graft-versus-host disease (GVHD) that precluded further administration of ipilimumab was observed in 4 patients (14%). No responses that met formal response criteria occurred in patients who received a dose of 3 mg per kilogram. Among 22 patients who received a dose of 10 mg per kilogram, 5 (23%) had a complete response, 2 (9%) had a partial response, and 6 (27%) had decreased tumor burden. Complete responses occurred in 4 patients with extramedullary acute myeloid leukemia and 1 patient with the myelodysplastic syndrome developing into acute myeloid leukemia. Four patients had a durable response for more than 1 year. Responses were associated with in situ infiltration of cytotoxic CD8+ T cells, decreased activation of regulatory T cells, and expansion of subpopulations of effector T cells in the blood. CONCLUSIONS: Our early-phase data showed that administration of ipilimumab was feasible in patients with recurrent hematologic cancers after allogeneic HSCT, although immune-mediated toxic effects and GVHD occurred. Durable responses were observed in association with several histologic subtypes of these cancers, including extramedullary acute myeloid leukemia. (Funded by the National Institutes of Health and others; ClinicalTrials.gov number, NCT01822509.).

Calcium intake and risk of colorectal cancer according to expression status of calcium-sensing receptor (CASR).

OBJECTIVE: Although evidence suggests an inverse association between calcium intake and the risk of colorectal cancer, the mechanisms remain unclear. The calcium-sensing receptor (CASR) is expressed abundantly in normal colonic epithelium and may influence carcinogenesis. We hypothesized that calcium intake might be associated with lower risk of CASR-positive, but not CASR-negative, colorectal cancer. DESIGN: We assessed tumour CASR protein expression using immunohistochemistry in 779 incident colon and rectal cancer cases that developed among 136 249 individuals in the Nurses' Health Study and Health Professionals Follow-Up Study. Duplication method Cox proportional hazards regression analysis was used to assess associations of calcium intake with incidence of colorectal adenocarcinoma subtypes by CASR status. RESULTS: Total calcium intake was inversely associated with the risk of developing colorectal cancer (ptrend=0.01, comparing ≥1200 vs <600 mg/day: multivariable HR=0.75, 95% CI 0.60 to 0.95). For the same comparison, higher total calcium intake was associated with a lower risk of CASR-positive tumours (ptrend=0.003, multivariable HR=0.67, 95% CI 0.51 to 0.86) but not with CASR-negative tumours (ptrend=0.67, multivariable HR=1.15, 95% CI 0.75 to 1.78; pheterogeneity=0.06 between the CASR subtypes). The stronger inverse associations of calcium intake with CASR-positive but not CASR-negative tumours generally appeared consistent regardless of sex, tumour location and source of calcium. CONCLUSIONS: Our molecular pathological epidemiology data suggest a causal relationship between higher calcium intake and lower colorectal cancer risk, and a potential role of CASR in mediating antineoplastic effect of calcium.

AKT1low quiescent cancer cells persist after neoadjuvant chemotherapy in triple negative breast cancer.

BACKGROUND: Absence of pathologic complete response (pCR) to neoadjuvant chemotherapy (NACT) correlates with poor long-term survival in patients with triple negative breast cancer (TNBC). These incomplete treatment responses are likely determined by mechanisms that enable cancer cells to resist being killed. However, the detailed characterization of a drug-resistant cancer cell state in residual TNBC tissue after NACT has remained elusive. AKT1low quiescent cancer cells (QCCs) are a quiescent, epigenetically plastic, and chemotherapy-resistant subpopulation initially identified in experimental cancer models. Here, we asked whether QCCs exist in primary tumors from patients with TNBC and persist after treatment with NACT. METHODS: We obtained pre-treatment biopsy, post-treatment mastectomy, and metastatic specimens from a retrospective cohort of TNBC patients treated with NACT at Massachusetts General Hospital (n = 25). Using quantitative automated immunofluorescence microscopy, QCCs were identified as AKTlow/H3K9me2low/HES1high cancer cells using prespecified immunofluorescence intensity thresholds. QCCs were represented in 2D and 3D digital tumor maps and QCC percentage (QCC-P) and QCC cluster index (QCC-CI) were determined for each sample. RESULTS: We showed that QCCs exist as non-random and heterogeneously distributed clusters within primary breast tumors. In addition, these QCC clusters persist after treatment with multi-agent, multi-cycle, neoadjuvant chemotherapy in both residual primary tumors and nodal and distant metastases in patients with triple negative breast cancer. CONCLUSIONS: These first-in-human data potentially qualify AKT1low quiescent cancer cells as a non-genetic cell state that persists after neoadjuvant chemotherapy in triple negative breast cancer patients and warrants further study.

Tumor expression of calcium sensing receptor and colorectal cancer survival: Results from the nurses' health study and health professionals follow-up study.

Although experimental evidence suggests calcium-sensing receptor (CASR) as a tumor-suppressor, the prognostic role of tumor CASR expression in colorectal carcinoma remains unclear. We hypothesized that higher tumor CASR expression might be associated with improved survival among colorectal cancer patients. We evaluated tumor expression levels of CASR by immunohistochemistry in 809 incident colorectal cancer patients within the Nurses' Health Study and the Health Professionals Follow-up Study. We used Cox proportional hazards regression models to estimate multivariable hazard ratio (HR) for the association of tumor CASR expression with colorectal cancer-specific and all-cause mortality. We adjusted for potential confounders including tumor biomarkers such as microsatellite instability, CpG island methylator phenotype, LINE-1 methylation level, expressions of PTGS2, VDR and CTNNB1 and mutations of KRAS, BRAF and PIK3CA. There were 240 colorectal cancer-specific deaths and 427 all-cause deaths. The median follow-up of censored patients was 10.8 years (interquartile range: 7.2, 15.1). Compared with patients with no or weak expression of CASR, the multivariable HRs for colorectal cancer-specific mortality were 0.80 [95% confidence interval (CI): 0.55-1.16] in patients with moderate CASR expression and 0.50 (95% CI: 0.32-0.79) in patients with intense CASR expression (p-trend = 0.003). The corresponding HRs for overall mortality were 0.85 (0.64-1.13) and 0.81 (0.58-1.12), respectively. Higher tumor CASR expression was associated with a lower risk of colorectal cancer-specific mortality. This finding needs further confirmation and if confirmed, may lead to better understanding of the role of CASR in colorectal cancer progression.

Differential Expression of PD-L1 in High Grade T1 vs Muscle Invasive Bladder Carcinoma and its Prognostic Implications.

PURPOSE: PD-L1 is expressed on tumor cells and tumor immune cell infiltrates. In metastatic bladder cancer increased tumor immune cell infiltrate PD-L1 positivity correlated with better overall survival. However, to our knowledge in high grade T1 bladder tumors positivity on tumor cells and tumor immune cell infiltrates, and correlation with outcomes or pathological features remain unknown. MATERIALS AND METHODS: Formalin fixed, paraffin embedded tumor samples from 140 patients with clinically annotated, high grade T1 bladder tumors were retrieved. All patients were initially diagnosed with high grade T1 bladder tumors by transurethral resection, subsequently received bacillus Calmette-Guérin and had a median followup of 7.4 years. PD-L1 positivity on initial transurethral resection was evaluated by immunohistochemistry using a mouse monoclonal antiPD-L1 antibody (405.9A11). Tumor cell PD-L1 positivity was defined as staining of 5% of the tumor cell membrane. Tumor immune cell infiltrate PD-L1 positivity was scored based on the extent of infiltrate and the percent of positive cells. The Fisher exact test was used to assess associations of PD-L1 positivity with disease outcomes, carcinoma in situ presence and the difference between high grade T1 bladder tumors and muscle invasive bladder cancer. RESULTS: Among 140 patients with high grade T1 bladder tumors tumor cells and tumor immune cell infiltrate PD-L1 positivity was seen in 6 (4%) and 48 (34.3%), respectively. In a subset of 106 patients with adequate followup PD-L1 positivity did not correlate with disease outcomes on tumor cells (p = 0.3) or on tumor immune cell infiltrates (p = 0.47). PD-L1 positivity also did not correlate with the presence of carcinoma in situ. Tumor cell PD-L1 positivity was significantly less in high grade T1 bladder tumors than in muscle invasive bladder cancer (p <0.001). CONCLUSIONS: PD-L1 is widely expressed on tumor immune cell infiltrates but not on tumor cells in high grade T1 bladder tumors. We did not find a correlation between PD-L1 positivity and outcomes or carcinoma in situ presence. Tumor cell PD-L1 positivity is significantly lower in high grade T1 bladder tumors than in muscle invasive bladder cancer.

Erlotinib, cabozantinib, or erlotinib plus cabozantinib as second-line or third-line treatment of patients with EGFR wild-type advanced non-small-cell lung cancer (ECOG-ACRIN 1512): a randomised, controlled, open-label, multicentre, phase 2 trial.

BACKGROUND: Erlotinib is approved for the treatment of all patients with advanced non-small-cell lung cancer (NSCLC), but is most active in the treatment of EGFR mutant NSCLC. Cabozantinib, a small molecule tyrosine kinase inhibitor, targets MET, VEGFR, RET, ROS1, and AXL, which are implicated in lung cancer tumorigenesis. We compared the efficacy of cabozantinib alone or in combination with erlotinib versus erlotinib alone in patients with EGFR wild-type NSCLC. METHODS: This three group, randomised, controlled, open-label, multicentre, phase 2 trial was done in 37 academic and community oncology practices in the USA. Patients were eligible if they had received one or two previous treatments for advanced non-squamous, EGFR wild-type, NSCLC. Patients were stratified by performance status and line of therapy, and randomly assigned using permuted blocks within strata to receive open-label oral daily dosing of erlotinib (150 mg), cabozantinib (60 mg), or erlotinib (150 mg) and cabozantinib (40 mg). Imaging was done every 8 weeks. At the time of radiographic progression, there was optional crossover for patients in either single-drug group to receive combination treatment. The primary endpoint was to compare progression-free survival in patients given erlotinib alone versus cabozantinib alone, and in patients given erlotinib alone versus the combination of erlotinib plus cabozantinib. We assessed the primary endpoint in the per-protocol population, which was defined as all patients who were eligible, randomly assigned, and received at least one dose of treatment. The safety analysis population included all patients who received study treatment irrespective of eligibility. This trial is registered with ClinicalTrials.gov, number NCT01708954. FINDINGS: Between Feb 7, 2013, and July 1, 2014, we enrolled and randomly assigned 42 patients to erlotinib treatment, 40 patients to cabozantinib treatment, and 43 patients to erlotinib plus cabozantinib treatment, of whom 111 (89%) in total were included in the primary analysis (erlotinib [n=38], cabozantinib [n=38], erlotinib plus cabozantinib [n=35]). Compared with erlotinib alone (median 1·8 months [95% CI 1·7-2·2]), progression-free survival was significantly improved in the cabozantinib group (4·3 months [3·6-7·4]; hazard ratio [HR] 0·39, 80% CI 0·27-0·55; one-sided p=0·0003) and in the erlotinib plus cabozantinib group (4·7 months [2·4-7·4]; HR 0·37, 0·25-0·53; one-sided p=0·0003). Among participants included in the safety analysis of the erlotinib (n=40), cabozantinib (n=40), and erlotinib plus cabozantinib (n=39) groups, the most common grade 3 or 4 adverse events were diarrhoea (three [8%] cases in the erlotinib group vs three [8%] in the cabozantinib group vs 11 [28%] in the erlotinib plus cabozantinib group), hypertension (none vs ten [25%] vs one [3%]), fatigue (five [13%] vs six [15%] vs six [15%]), oral mucositis (none vs four [10%] vs one [3%]), and thromboembolic event (none vs three [8%] vs two [5%]). One death due to respiratory failure occurred in the cabozantinib group, deemed possibly related to either drug, and one death due to pneumonitis occurred in the erlotinib plus cabozantinib group, deemed related to either drug or the combination. INTERPRETATION: Despite its small sample size, this trial showed that, in patients with EGFR wild-type NSCLC, cabozantinib alone or combined with erlotinib has clinically meaningful, superior efficacy to that of erlotinib alone, with additional toxicity that was generally manageable. Cabozantinib-based regimens are promising for further investigation in this patient population. FUNDING: ECOG-ACRIN Cancer Research Group, National Cancer Institute of the National Institutes of Health.

Association of tumour microRNA profiling with outcomes in patients with advanced urothelial carcinoma receiving first-line platinum-based chemotherapy.

BACKGROUND: Tumour expression of selected microRNAs (miRs) correlates with cisplatin efficacy in multiple cancers. We investigated the role of selected miRs in patients receiving cisplatin-based therapy for advanced urothelial carcinoma (UC). METHODS: RNA was extracted from formalin-fixed paraffin-embedded tumour from 83 advanced UC patients who received cisplatin. A miR panel based on relevance for platinum sensitivity and UC was studied by quantitative reverse transcription quantitative PCR (RT-qPCR). Association of progression-free survival (PFS) with miR expression was analysed using cox regression. Selected TFs were chosen by association with the panel of miRs using the Transcription Regulation algorithm (GeneGo MetaCore+MetaDrug version 6.23 build 67496). Bladder cancer (BC) cell lines were used to investigate the previously described role of miR-21 mediating cisplatin sensitivity. RESULTS: The 83 patients had a median PFS of 8 months. In multivariate analysis, higher levels of E2F1 (P=0.01, HR: 1.95 (1.14, 3.33)), miR-21 (P=0.01, HR: 2.01 (1.17, 3.45)) and miR-372 (P=0.05, HR: 1.70 (1.00, 2.89)) were associated with a shorter PFS. In the 8 BC cell lines, miR-21 was not shown to be necessary nor sufficient for modulating cisplatin sensitivity. CONCLUSIONS: In metastatic UC patients treated with cisplatin-based therapy, high primary tumour levels of E2F1, miR-21 and miR-372 are associated with poor PFS independent of clinical prognostic factors. The in vitro study could not confirm miR-21 levels role in modulating platinum sensitivity.

Hallmarks of Resistance to Immune-Checkpoint Inhibitors.

Immune-checkpoint inhibitors (ICI), although revolutionary in improving long-term survival outcomes, are mostly effective in patients with immune-responsive tumors. Most patients with cancer either do not respond to ICIs at all or experience disease progression after an initial period of response. Treatment resistance to ICIs remains a major challenge and defines the biggest unmet medical need in oncology worldwide. In a collaborative workshop, thought leaders from academic, biopharma, and nonprofit sectors convened to outline a resistance framework to support and guide future immune-resistance research. Here, we explore the initial part of our effort by collating seminal discoveries through the lens of known biological processes. We highlight eight biological processes and refer to them as immune resistance nodes. We examine the seminal discoveries that define each immune resistance node and pose critical questions, which, if answered, would greatly expand our notion of immune resistance. Ultimately, the expansion and application of this work calls for the integration of multiomic high-dimensional analyses from patient-level data to produce a map of resistance phenotypes that can be utilized to guide effective drug development and improved patient outcomes.

Transcriptomic analysis of micropapillary high grade T1 urothelial bladder cancer.

No consensus currently exist on the optimal treatment of patients with high-risk nonmuscle invasive (HGT1) micropapillary variant of bladder cancer (MPBC). Transcripsome analysis may allow stratification of MPBC-HGT1 enabling prediction of recurrence and guide therapeutic management for individual patients. Whole transcriptome RNA-Sequencing of tumors from 23 patients with MPBC-HGT1 and 64 conventional urothelial carcinomas (cUC) (reference set) was performed. Differentially expressed genes between MPBC-HGT1 and cUC-HGT1 were explored. Cox proportional hazard models and Kapplan-Meier methods were used to assess the relation between time to progression (TTP) and individual gene expression adjusting for clinical covariates. Over 3000 genes were differentially expressed in MPBC-HGT1 as compared with cUC-HGT1 and a 26-gene signature is characteristic of MPBC within HGT1. A set of three genes; CD36, FAPB3 and RAETE1; were significantly associated with TTP. High expression of FABP3 and CD36 were associated with shorter TTP (p = 0.045 and p = 0.08) as was low expression of RAET1E (p = 0.01). Our study suggest that a 26-gene signature can define MPBC-HGT1 within conventional urothelial carcinomas. A prognostic risk index of three genes (FABP3, CD36 and RAET1E) was found to be associated with shorter TTP and may help classify a group of patients with MPBC-HGT1 with high-risk of early progression. These observations might have implications in terms of radical cystectomy recommendation in MPBC patients.

Multiplex Immunofluorescence in Formalin-Fixed Paraffin-Embedded Tumor Tissue to Identify Single-Cell-Level PI3K Pathway Activation.

PURPOSE: Identifying cancers with high PI3K pathway activity is critical for treatment selection and eligibility into clinical trials of PI3K inhibitors. Assessments of tumor signaling pathway activity need to consider intratumoral heterogeneity and multiple regulatory nodes. EXPERIMENTAL DESIGN: We established a novel, mechanistically informed approach to assessing tumor signaling pathways by quantifying single-cell-level multiplex immunofluorescence using custom algorithms. In a proof-of-concept study, we stained archival formalin-fixed, paraffin-embedded (FFPE) tissue from patients with primary prostate cancer in two prospective cohort studies, the Health Professionals Follow-up Study and the Physicians' Health Study. PTEN, stathmin, and phospho-S6 were quantified on 14 tissue microarrays as indicators of PI3K activation to derive cell-level PI3K scores. RESULTS: In 1,001 men, 988,254 tumor cells were assessed (median, 743 per tumor; interquartile range, 290-1,377). PI3K scores were higher in tumors with PTEN loss scored by a pathologist, higher Gleason grade, and a new, validated bulk PI3K transcriptional signature. Unsupervised machine-learning approaches resulted in similar clustering. Within-tumor heterogeneity in cell-level PI3K scores was high. During long-term follow-up (median, 15.3 years), rates of progression to metastases and death from prostate cancer were twice as high in the highest quartile of PI3K activation compared with the lowest quartile (hazard ratio, 2.04; 95% confidence interval, 1.13-3.68). CONCLUSIONS: Our novel pathway-focused approach to quantifying single-cell-level immunofluorescence in FFPE tissue identifies prostate tumors with PI3K pathway activation that are more aggressive and may respond to pathway inhibitors.

Genomic Predictors of Good Outcome, Recurrence, or Progression in High-Grade T1 Non-Muscle-Invasive Bladder Cancer.

High-grade T1 (HGT1) bladder cancer is the highest risk subtype of non-muscle-invasive bladder cancer with unpredictable outcome and poorly understood risk factors. Here, we examined the association of somatic mutation profiles with nonrecurrent disease (GO, good outcome), recurrence (R), or progression (PD) in a cohort of HGT1 patients. Exome sequencing was performed on 62 HGT1 and 15 matched normal tissue samples. Both tumor only (TO) and paired analyses were performed, focusing on 95 genes known to be mutated in bladder cancer. Somatic mutations, copy-number alterations, mutation load, and mutation signatures were studied. Thirty-three GO, 10 R, 18 PD, and 1 unknown outcome patients were analyzed. Tumor mutational burden (TMB) was similar to muscle-invasive disease and was highest in GO, intermediate in PD, and lowest in R patients (P = 0.017). DNA damage response gene mutations were associated with higher TMB (P < 0.0001) and GO (P = 0.003). ERCC2 and BRCA2 mutations were associated with GO. TP53, ATM, ARID1A, AHR, and SMARCB1 mutations were more frequent in PD. Focal copy-number gain in CCNE1 and CDKN2A deletion was enriched in PD or R (P = 0.047; P = 0.06). APOBEC (46%) and COSMIC5 (34%) signatures were most frequent. APOBEC-A and ERCC2 mutant tumors (COSMIC5) were associated with GO (P = 0.047; P = 0.0002). pT1b microstaging was associated with a genomic cluster (P = 0.05) with focal amplifications of E2F3/SOX4, PVRL4, CCNE1, and TP53 mutations. Findings were validated using external public datasets. These findings require confirmation but suggest that management of HGT1 bladder cancer may be improved via molecular characterization to predict outcome. SIGNIFICANCE: Detailed genetic analyses of HGT1 bladder tumors identify features that correlate with outcome, e.g., high mutational burden, ERCC2 mutations, and high APOBEC-A/ERCC2 mutation signatures were associated with good outcome.

Defining tumor resistance to PD-1 pathway blockade: recommendations from the first meeting of the SITC Immunotherapy Resistance Taskforce.

As the field of cancer immunotherapy continues to advance at a fast pace, treatment approaches and drug development are evolving rapidly to maximize patient benefit. New agents are commonly evaluated for activity in patients who had previously received a programmed death receptor 1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitor as standard of care or in an investigational study. However, because of the kinetics and patterns of response to PD-1/PD-L1 blockade, and the lack of consistency in the clinical definitions of resistance to therapy, the design of clinical trials of new agents and interpretation of results remains an important challenge. To address this unmet need, the Society for Immunotherapy of Cancer convened a multistakeholder taskforce-consisting of experts in cancer immunotherapy from academia, industry, and government-to generate consensus clinical definitions for resistance to PD-(L)1 inhibitors in three distinct scenarios: primary resistance, secondary resistance, and progression after treatment discontinuation. The taskforce generated consensus on several key issues such as the timeframes that delineate each type of resistance, the necessity for confirmatory scans, and identified caveats for each specific resistance classification. The goal of this effort is to provide guidance for clinical trial design and to support analyses of emerging molecular and cellular data surrounding mechanisms of resistance.

Loss of PTEN Expression Detected by Fluorescence Immunohistochemistry Predicts Lethal Prostate Cancer in Men Treated with Prostatectomy.

BACKGROUND: PTEN deletion is associated with relapse after therapy for localized prostate cancer. There are limited data on PTEN loss as detected by immunohistochemistry (IHC) and the risk of lethal disease after surgery. OBJECTIVE: To determine whether PTEN loss as detected by quantitative fluorescence IHC (FIHC) predicts lethal disease outcomes after surgery for prostate cancer. DESIGN, SETTING AND PARTICIPANTS: We used formalin-fixed, paraffin-embedded radical prostatectomy specimens to construct tissue microarrays and perform dual FIHC for PTEN and AMACR for masking tumor epithelium, plus semi-quantitative multispectral imaging analysis. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The association of PTEN status analyzed continuously and dichotomously (low [expression in the lowest quartile] vs higher [expression >lowest quartile]) with disease outcomes (metastasis and death) was assessed with adjustment for age, Gleason score, and stage in multivariable analyses. The prognostic ability of PTEN was assessed using logistic regression models. RESULTS AND LIMITATIONS: Low PTEN expression was associated with a higher risk of metastatic disease as both a continuous (hazard ratio [HR] 1.49, 95% confidence interval [CI] 1.14-1.92; p<0.003) and dichotomous (HR 1.92, 95% CI 1.02-3.63; p=0.04) variable. A significant association between low PTEN expression and poorer overall survival was observed (continuous: HR 1.89, 95% CI 1.37-2.63; p<0.001; dichotomous: HR 2.66, 95% CI 1.34-5.28; p=0.005). Addition of PTEN status to clinicopathologic factors (age, Gleason score, and stage) incrementally improved a prognostic model assessing 10-yr outcomes for metastatic disease (area under the curve [AUC] 0.76 vs 0.80) and death (AUC 0.70 vs 0.75). CONCLUSIONS: Low PTEN expression detected by FIHC in primary prostate cancer is an independent prognostic biomarker for metastatic disease and death after definitive therapy. FIHC for PTEN is a viable clinical diagnostic assay in this context. PATIENT SUMMARY: We looked at loss of the PTEN protein in prostate tumors from men treated with surgery. Men with PTEN loss were at higher risk of metastasis and death. Assessing PTEN status may be useful in better determination of the risk of poorer outcomes.