Direct transposition of native DNA for sensitive multimodal single-molecule sequencing.

Concurrent readout of sequence and base modifications from long unamplified DNA templates by Pacific Biosciences of California (PacBio) single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90-99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000-50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.

A germline point mutation in the MYC-FBW7 phosphodegron initiates hematopoietic malignancies.

Oncogenic activation of MYC in cancers predominantly involves increased transcription rather than coding region mutations. However, MYC-dependent lymphomas frequently acquire point mutations in the MYC phosphodegron, including at threonine 58 (T58), where phosphorylation permits binding via the FBW7 ubiquitin ligase triggering MYC degradation. To understand how T58 phosphorylation functions in normal cell physiology, we introduced an alanine mutation at T58 (T58A) into the endogenous c-Myc locus in the mouse germline. While MYC-T58A mice develop normally, lymphomas and myeloid leukemias emerge in ∼60% of adult homozygous T58A mice. We found that primitive hematopoietic progenitor cells from MYC-T58A mice exhibit aberrant self-renewal normally associated with hematopoietic stem cells (HSCs) and up-regulate a subset of MYC target genes important in maintaining stem/progenitor cell balance. In lymphocytes, genomic occupancy by MYC-T58A was increased at all promoters compared with WT MYC, while genes differentially expressed in a T58A-dependent manner were significantly more proximal to MYC-bound enhancers. MYC-T58A lymphocyte progenitors exhibited metabolic alterations and decreased activation of inflammatory and apoptotic pathways. Our data demonstrate that a single point mutation stabilizing MYC is sufficient to skew target gene expression, producing a profound gain of function in multipotential hematopoietic progenitors associated with self-renewal and initiation of lymphomas and leukemias.

A Germline Point Mutation in the MYC-FBW7 Phosphodegron Initiates Hematopoietic Malignancies.

Oncogenic activation of MYC in cancers predominantly involves increased transcription rather than coding region mutations. However, MYC-dependent lymphomas frequently contain point mutations in the MYC phospho-degron, including at threonine-58 (T58), where phosphorylation permits binding by the FBW7 ubiquitin ligase triggering MYC degradation. To understand how T58 phosphorylation functions in normal cell physiology, we introduced an alanine mutation at T58 (T58A) into the endogenous c-Myc locus in the mouse germline. While MYC-T58A mice develop normally, lymphomas and myeloid leukemias emerge in ~60% of adult homozygous T58A mice. We find that primitive hematopoietic progenitor cells from MYC-T58A mice exhibit aberrant self-renewal normally associated with hematopoietic stem cells (HSCs) and upregulate a subset of Myc target genes important in maintaining stem/progenitor cell balance. Genomic occupancy by MYC-T58A was increased at all promoters, compared to WT MYC, while genes differentially expressed in a T58A-dependent manner were significantly more proximal to MYC-bound enhancers. MYC-T58A lymphocyte progenitors exhibited metabolic alterations and decreased activation of inflammatory and apoptotic pathways. Our data demonstrate that a single point mutation in Myc is sufficient to produce a profound gain of function in multipotential hematopoietic progenitors associated with self-renewal and initiation of lymphomas and leukemias.

Involvement of endoplasmic reticulum and histone proteins in immunomodulation by TLR4-interacting SPA4 peptide against Escherichia coli.

Pulmonary host defense is critical for the control of lung infection and inflammation. An increased expression and activity of Toll-like receptor 4 (TLR4) induce phagocytic uptake/clearance and inflammation against Gram-negative bacteria. In this study, we addressed the mechanistic aspect of the immunomodulatory activity of the TLR4-interacting SPA4 peptide (amino acid sequence GDFRYSDGTPVNYTNWYRGE) against Escherichia coli. Binding of the SPA4 peptide to bacteria and direct anti-bacterial effects were investigated using flow cytometric, microscopic, and bacteriological methods. The bacterial uptake and inflammatory cytokine response were studied in dendritic cells expressing endogenous basal level of TLR4 or overexpressing TLR4. The subcellular distribution and co-localization of TLR4 and bacteria were investigated by immunocytochemistry. Furthermore, we studied the cellular expression and co-localization of endoplasmic reticulum (ER) molecules (calnexin and ER membrane protein complex subunit 1; EMC1) with lysosomal-associated membrane protein 1 (LAMP1) in cells infected with E. coli and treated with the SPA4 peptide. Simultaneously, the expression of histone H2A protein was quantitated by immunoblotting. Our results demonstrate no binding or direct killing of the bacteria by SPA4 peptide. Instead, it induces the uptake and localization of E. coli in the phagolysosomes for lysis and simultaneously suppresses the secreted levels of TNF-α. Overexpression of TLR4 further augments the pro-phagocytic and anti-inflammatory activity of SPA4 peptide. A time-dependent change in subcellular distribution of TLR4 and an increased co-localization of TLR4 with E. coli in SPA4 peptide-treated cells suggest an enhanced recognition and internalization of bacteria in conjugation with TLR4. Furthermore, an increased co-localization of calnexin and EMC1 with LAMP1 indicates the involvement of ER in pro-phagocytic activity of SPA4 peptide. Simultaneous reduction in secreted amounts of TNF-α coincides with suppressed histone H2A protein expression in the SPA4 peptide-treated cells. These results provide initial insights into the plausible role of ER and histones in the TLR4-immunomodulatory activity of SPA4 peptide against Gram-negative bacteria.

Nucleosome density shapes kilobase-scale regulation by a mammalian chromatin remodeler.

Nearly all essential nuclear processes act on DNA packaged into arrays of nucleosomes. However, our understanding of how these processes (for example, DNA replication, RNA transcription, chromatin extrusion and nucleosome remodeling) occur on individual chromatin arrays remains unresolved. Here, to address this deficit, we present SAMOSA-ChAAT: a massively multiplex single-molecule footprinting approach to map the primary structure of individual, reconstituted chromatin templates subject to virtually any chromatin-associated reaction. We apply this method to distinguish between competing models for chromatin remodeling by the essential imitation switch (ISWI) ATPase SNF2h: nucleosome-density-dependent spacing versus fixed-linker-length nucleosome clamping. First, we perform in vivo single-molecule nucleosome footprinting in murine embryonic stem cells, to discover that ISWI-catalyzed nucleosome spacing correlates with the underlying nucleosome density of specific epigenomic domains. To establish causality, we apply SAMOSA-ChAAT to quantify the activities of ISWI ATPase SNF2h and its parent complex ACF on reconstituted nucleosomal arrays of varying nucleosome density, at single-molecule resolution. We demonstrate that ISWI remodelers operate as density-dependent, length-sensing nucleosome sliders, whose ability to program DNA accessibility is dictated by single-molecule nucleosome density. We propose that the long-observed, context-specific regulatory effects of ISWI complexes can be explained in part by the sensing of nucleosome density within epigenomic domains. More generally, our approach promises molecule-precise views of the essential processes that shape nuclear physiology.

Bladder-Sparing Treatment With Radical Dose Radiotherapy Is an Effective Alternative to Radical Cystectomy in Patients With Clinically Node-Positive Nonmetastatic Bladder Cancer.

PURPOSE: Bladder-sparing trimodal therapy (TMT) is an alternative to radical cystectomy (RC) according to international guidelines. However, there are limited data to guide management of nonmetastatic clinically node-positive bladder cancer (cN+ M0 BCa). We performed a multicenter retrospective analysis of survival outcomes in node-positive patients to inform practice. METHODS: Data from patients diagnosed with cN+ M0 BCa were collected from participating UK Oncology centers offering both TMT and RC. Overall survival (OS) and progression-free survival (PFS) outcomes were collected with details of treatment and clinical factors. RESULTS: A total of 287 patients with cN+ M0 BCa were included in the survival analysis. Median OS across all patients was 1.55 years (95% CI, 1.35 to 1.82 years). Receiving radical treatments was associated with improved OS (hazard ratio [HR], 0.32; 95% CI, 0.23 to 0.44; P < .001) compared with receiving palliative treatment. Radically treated patients (n = 163) received RC (n = 76) or radical dose radiotherapy (RT, n = 87); choice of radical treatment showed no association with OS (HR, 0.94; 95% CI, 0.63 to 1.41; P = .76) or PFS (HR, 0.74; 95% CI, 0.50 to 1.08; P = .12) on multivariable analysis. CONCLUSION: Patient cohorts with cN+ M0 BCa had equivalent survival outcomes whether treated with surgery or radical RT. Given the known morbidities of RC-in a patient group with poor survival-this study confirms that bladder-sparing TMT treatment should be a treatment option available to all patients with cN+ M0 BCa.

Dendritic cell therapy augments antitumor immunity triggered by CDK4/6 inhibition and immune checkpoint blockade by unleashing systemic CD4 T-cell responses.

BACKGROUND: Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) combined with endocrine therapy are a mainstay treatment for hormone receptor-positive breast cancer. While their principal mechanism is inhibition of cancer cell proliferation, preclinical and clinical evidence suggests that CDK4/6i can also promote antitumor T-cell responses. However, this pro-immunogenic property is yet to be successfully harnessed in the clinic, as combining CDK4/6i with immune checkpoint blockade (ICB) has not shown a definitive benefit in patients. METHOD: We performed an in-depth analysis of the changes in the tumor immune microenvironment and systemic immune modulation associated with CDK4/6i treatment in muring breast cancer models and in patients with breast cancer using high dimensional flow cytometry and RNA sequencing. Gain and loss of function in vivo experiments employing cell transfer and depletion antibody were performed to uncover immune cell populations critical for CDK4/6i-mediated stimulation of antitumor immunity. RESULTS: We found that loss of dendritic cells (DCs) within the tumor microenvironment resulting from CDK4/6 inhibition in bone marrow progenitors is a major factor limiting antitumor immunity after CDK4/6i and ICB. Consequently, restoration of DC compartment by adoptively transferring ex vivo differentiated DCs to mice treated with CDK4/6i and ICB therapy enabled robust tumor inhibition. Mechanistically, the addition of DCs promoted the induction of tumor-localized and systemic CD4 T-cell responses in mice receiving CDK4/6i-ICB-DC combination therapy, as characterized by enrichment of programmed cell death protein-1-negative T helper (Th)1 and Th2 cells with an activated phenotype. CD4 T-cell depletion abrogated the antitumor benefit of CDK4/6i-ICB-DC combination, with outgrowing tumors displaying an increased proportion of terminally exhausted CD8 T cells. CONCLUSIONS: Our findings suggest that CDK4/6i-mediated DC suppression limits CD4 T-cell responses essential for the sustained activity of CD8 T cells and tumor inhibition. Furthermore, they imply that restoring DC-CD4 T-cell crosstalk via DC transfer enables effective breast cancer immunity in response to CDK4/6i and ICB treatment.

Cost-effectiveness model of renal cell carcinoma (RCC) surveillance in hereditary leiomyomatosis and renal cell carcinoma (HLRCC).

PURPOSE: To determine the cost-effectiveness of annual renal imaging surveillance (RIS) in hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC is associated with a 21% risk to age 70 years of RCC. Presentations with advanced renal cell cancer (RCC) are associated with poor outcomes whereas RIS detects early-stage RCC; however, evidence for the cost-effectiveness of RIS is lacking. METHODS: We developed a decision-analytic model to compare, at different age starting points (11 years, 18 years, 40 years, 60 years), the costs and benefits of lifetime contrast-enhanced renal MRI surveillance (CERMRIS) vs no surveillance in HLRCC. Benefits were measured in life-years gained (LYG), quality-adjusted life years (QALYs) and costs in British Pounds Sterling (GBP). Net monetary benefit (NMB) was calculated using a cost-effectiveness threshold of £20 000/QALY. One-way sensitivity and probabilistic analyses were also performed. RESULTS: In the base-case 11-year age cohort, surveillance was cost-effective (Incremental_NMB=£3522 (95% CI -£2747 to £7652); Incremental_LYG=1.25 (95% CI 0.30 to 1.86); Incremental_QALYs=0.29 (95% CI 0.07 to 0.43)] at an additional mean discounted cost of £2185/patient (95% CI £430 to £4144). Surveillance was also cost-effective in other age cohorts and dominated a no surveillance strategy in the 40 year cohort [Incremental_NMB=£12 655 (95% CIs -£709 to £21 134); Incremental_LYG=1.52 (95% CI 0.30 to 2.26); Incremental_QALYs=0.58 (95% CI 0.12 to 0.87) with a cost saving of £965/patient (95% CI -£4202 to £2652). CONCLUSION: Annual CERMRI in HLRCC is cost-effective across age groups modelled.

Radiotherapy to the prostate for men with metastatic prostate cancer in the UK and Switzerland: Long-term results from the STAMPEDE randomised controlled trial.

BACKGROUND: STAMPEDE has previously reported that radiotherapy (RT) to the prostate improved overall survival (OS) for patients with newly diagnosed prostate cancer with low metastatic burden, but not those with high-burden disease. In this final analysis, we report long-term findings on the primary outcome measure of OS and on the secondary outcome measures of symptomatic local events, RT toxicity events, and quality of life (QoL). METHODS AND FINDINGS: Patients were randomised at secondary care sites in the United Kingdom and Switzerland between January 2013 and September 2016, with 1:1 stratified allocation: 1,029 to standard of care (SOC) and 1,032 to SOC+RT. No masking of the treatment allocation was employed. A total of 1,939 had metastatic burden classifiable, with 42% low burden and 58% high burden, balanced by treatment allocation. Intention-to-treat (ITT) analyses used Cox regression and flexible parametric models (FPMs), adjusted for stratification factors age, nodal involvement, the World Health Organization (WHO) performance status, regular aspirin or nonsteroidal anti-inflammatory drug (NSAID) use, and planned docetaxel use. QoL in the first 2 years on trial was assessed using prospectively collected patient responses to QLQ-30 questionnaire. Patients were followed for a median of 61.3 months. Prostate RT improved OS in patients with low, but not high, metastatic burden (respectively: 202 deaths in SOC versus 156 in SOC+RT, hazard ratio (HR) = 0·64, 95% CI 0.52, 0.79, p < 0.001; 375 SOC versus 386 SOC+RT, HR = 1.11, 95% CI 0.96, 1.28, p = 0·164; interaction p < 0.001). No evidence of difference in time to symptomatic local events was found. There was no evidence of difference in Global QoL or QLQ-30 Summary Score. Long-term urinary toxicity of grade 3 or worse was reported for 10 SOC and 10 SOC+RT; long-term bowel toxicity of grade 3 or worse was reported for 15 and 11, respectively. CONCLUSIONS: Prostate RT improves OS, without detriment in QoL, in men with low-burden, newly diagnosed, metastatic prostate cancer, indicating that it should be recommended as a SOC. TRIAL REGISTRATION: ClinicalTrials.gov NCT00268476, ISRCTN.com ISRCTN78818544.

The glucose-sensing transcription factor MLX balances metabolism and stress to suppress apoptosis and maintain spermatogenesis.

Male germ cell (GC) production is a metabolically driven and apoptosis-prone process. Here, we show that the glucose-sensing transcription factor (TF) MAX-Like protein X (MLX) and its binding partner MondoA are both required for male fertility in the mouse, as well as survival of human tumor cells derived from the male germ line. Loss of Mlx results in altered metabolism as well as activation of multiple stress pathways and GC apoptosis in the testes. This is concomitant with dysregulation of the expression of male-specific GC transcripts and proteins. Our genomic and functional analyses identify loci directly bound by MLX involved in these processes, including metabolic targets, obligate components of male-specific GC development, and apoptotic effectors. These in vivo and in vitro studies implicate MLX and other members of the proximal MYC network, such as MNT, in regulation of metabolism and differentiation, as well as in suppression of intrinsic and extrinsic death signaling pathways in both spermatogenesis and male germ cell tumors (MGCTs).

The Role of Frozen Section Examination During Inguinal Exploration in Men with Inconclusive Testicular Tumors: A Systematic Review and Meta-analysis.

For inconclusive testicular tumors with negative tumor markers, frozen section examination (FSE) during inguinal exploration is recommended. However, FSE is time-consuming and therefore often not requested. Furthermore, the exact diagnostic benefit remains poorly defined. We performed a systematic review and meta-analysis summarizing 12 published studies and our own series of FSE in patients with inconclusive testicular tumors, resulting in a cohort of 1052 FSEs. FSE showed sensitivity of 99% and specificity of 96% with a positive predictive value of 98% and a negative predictive value of 97%. Most importantly, one-third of all testicular tumors investigated were correctly identified as being suitable for testis-sparing surgery and orchiectomy could be avoided. For patients with inconclusive testicular tumors, FSE is useful for deciding whether testis-sparing surgery is an option or whether radical orchiectomy should be performed. Thus, these patients should be optimally treated in institutions where FSE is available. PATIENT SUMMARY: We found that intraoperative examination of a frozen section is useful in deciding on whether the entire or only parts of the testicle can be removed. We conclude that frozen section examination should be offered to men with small testicular lesions and negative tumor markers.

Massively multiplex chemical transcriptomics at single-cell resolution.

High-throughput chemical screens typically use coarse assays such as cell survival, limiting what can be learned about mechanisms of action, off-target effects, and heterogeneous responses. Here, we introduce "sci-Plex," which uses "nuclear hashing" to quantify global transcriptional responses to thousands of independent perturbations at single-cell resolution. As a proof of concept, we applied sci-Plex to screen three cancer cell lines exposed to 188 compounds. In total, we profiled ~650,000 single-cell transcriptomes across ~5000 independent samples in one experiment. Our results reveal substantial intercellular heterogeneity in response to specific compounds, commonalities in response to families of compounds, and insight into differential properties within families. In particular, our results with histone deacetylase inhibitors support the view that chromatin acts as an important reservoir of acetate in cancer cells.

DC-HIL/Gpnmb Is a Negative Regulator of Tumor Response to Immune Checkpoint Inhibitors.

PURPOSE: Immune checkpoint inhibitors (ICI) benefit only a minority of treated patients with cancer. Identification of biomarkers distinguishing responders and nonresponders will improve management of patients with cancer. Because the DC-HIL checkpoint differs from the PD1 pathway in expression and inhibitory mechanisms, we examined whether DC-HIL expression regulates ICI responsiveness. EXPERIMENTAL DESIGN: Plasma samples were collected from patients with advanced non-small cell lung carcinoma (NSCLC) (n = 76) at baseline and/or follow-up after ICI monotherapy. Blood-soluble DC-HIL (sDC-HIL) was determined and analyzed for correlation with the early tumor response. To study the mechanisms, we measured effect of anti-DC-HIL versus anti-PDL1 mAb on growth of mouse tumor cells in experimentally metastatic lung. Influence of DC-HIL to anti-PDL1 treatment was assessed by changes in tumor response after deletion of host-DC-HIL gene, injection of DC-HIL-expressing myeloid-derived suppressor cells (MDSC), or induction of sDC-HIL expression. RESULTS: Nonresponders expressed significantly higher levels of baseline sDC-HIL levels than responders. Among patients (n = 28) for fluctuation with time, nonresponders (14/15 cases) showed increasing or persistently elevated levels. Responders (12/13) had decreasing or persistently low levels. Among various tumors, B16 melanoma exhibited resistance to anti-PDL1 but responded to anti-DC-HIL mAb. Using B16 melanoma and LL2 lung cancer, we showed that deletion of host-derived DC-HIL expression converted the resistant tumor to one responsive to anti-PDL1 mAb. The responsive state was reversed by infusion of DC-HIL+MDSC or induction of sDC-HIL expression. CONCLUSIONS: sDC-HIL in the blood and probably DC-HIL receptor expressed by MDSC play an important role in regulating response to ICI in advanced NSCLC.

Genomic Evaluation of Multiparametric Magnetic Resonance Imaging-visible and -nonvisible Lesions in Clinically Localised Prostate Cancer.

BACKGROUND: The prostate cancer (PCa) diagnostic pathway is undergoing a radical change with the introduction of multiparametric magnetic resonance imaging (mpMRI), genomic testing, and different prostate biopsy techniques. It has been proposed that these tests should be used in a sequential manner to optimise risk stratification. OBJECTIVE: To characterise the genomic, epigenomic, and transcriptomic features of mpMRI-visible and -nonvisible PCa in clinically localised disease. DESIGN, SETTING, AND PARTICIPANTS: Multicore analysis of fresh prostate tissue sampled immediately after radical prostatectomy was performed for intermediate- to high-risk PCa. INTERVENTION: Low-pass whole-genome, exome, methylation, and transcriptome profiling of patient tissue cores taken from microscopically benign and cancerous areas in the same prostate. Circulating free and germline DNA was assessed from the blood of five patients. OUTCOME MEASUREMENT AND STATISTICAL ANALYSIS: Correlations between preoperative mpMRI and genomic characteristics of tumour and benign prostate samples were assessed. Gene profiles for individual tumour cores were correlated with existing genomic classifiers currently used for prognostication. RESULTS AND LIMITATIONS: A total of 43 prostate cores (22 tumour and 21 benign) were profiled from six whole prostate glands. Of the 22 tumour cores, 16 were tumours visible and six were tumours nonvisible on mpMRI. Intratumour genomic, epigenomic, and transcriptomic heterogeneity was found within mpMRI-visible lesions. This could potentially lead to misclassification of patients using signatures based on copy number or RNA expression. Moreover, three of the six cores obtained from mpMRI-nonvisible tumours harboured one or more genetic alterations commonly observed in metastatic castration-resistant PCa. No circulating free DNA alterations were found. Limitations include the small cohort size and lack of follow-up. CONCLUSIONS: Our study supports the continued use of systematic prostate sampling in addition to mpMRI, as avoidance of systematic biopsies in patients with negative mpMRI may mean that clinically significant tumours harbouring genetic alterations commonly seen in metastatic PCa are missed. Furthermore, there is inconsistency in individual genomics when genomic classifiers are applied. PATIENT SUMMARY: Our study shows that tumour heterogeneity within prostate tumours visible on multiparametric magnetic resonance imaging (mpMRI) can lead to misclassification of patients if only one core is used for genomic analysis. In addition, some cancers that were missed by mpMRI had genomic aberrations that are commonly seen in advanced metastatic prostate cancer. Avoiding biopsies in mpMRI-negative cases may mean that such potentially lethal cancers are missed.

MRE11 as a Predictive Biomarker of Outcome After Radiation Therapy in Bladder Cancer.

PURPOSE: Organ-confined muscle-invasive bladder cancer is treated with cystectomy or bladder preservation techniques, including radiation therapy. There are currently no biomarkers to inform management decisions and aid patient choice. Previously we showed high levels of MRE11 protein, assessed by immunohistochemistry (IHC), predicted outcome after radiation therapy, but not cystectomy. Therefore, we sought to develop the MRE11 IHC assay for clinical use and define its relationship to clinical outcome in samples from 2 major clinical trials. METHODS AND MATERIALS: Samples from the BCON and BC2001 randomized controlled trials and a cystectomy cohort were stained using automated IHC methods and scored for MRE11 in 3 centers in the United Kingdom. RESULTS: Despite step-wise creation of scoring cards and standard operating procedures for staining and interpretation, there was poor intercenter scoring agreement (kappa, 0.32; 95% confidence interval, 0.17-0.47). No significant associations between MRE11 scores and cause-specific survival were identified in BCON (n = 132) and BC2001 (n = 221) samples. Reoptimized staining improved agreement between scores from BCON tissue microarrays (n = 116), but MRE11 expression was not prognostic for cause-specific survival. CONCLUSIONS: Manual IHC scoring of MRE11 was not validated as a reproducible biomarker of radiation-based bladder preservation success. There is a need for automated quantitative methods or a reassessment of how DNA-damage response relates to clinical outcomes.

Primary Mutational Landscape Linked with Pre-Docetaxel Lactate Dehydrogenase Levels Predicts Docetaxel Response in Metastatic Castrate-Resistant Prostate Cancer.

BACKGROUND: Docetaxel chemotherapy is a standard of care for metastatic castrate-resistant prostate cancer (mCRPC): 40-50% of patients achieve a biochemical response. However, there is a lack of response predictive biomarkers. OBJECTIVE: To assess lactate dehydrogenase (LDH) as a docetaxel response biomarker in mCRPC and to examine the association of LDH with genomic alterations in primary diagnostic biopsies. DESIGN, SETTING, AND PARTICIPANTS: Clinical and associated primary tumour-targeted next-generation sequencing data from matched training (n=150) and test (n=120) cohorts of progressive mCRPC patients receiving docetaxel therapy were analysed. Data were correlated with large-scale prostate cancer genomic datasets. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Prostate-specific antigen (PSA) response, radiographic response, biochemical progression-free survival (PFS), overall survival (OS), genomic analysis of primary biopsies, and genomic datasets (Memorial Sloan Kettering Cancer Center [MSKCC] and SU2C/PCF). RESULTS AND LIMITATIONS: Serum LDH ≥450U/l is a reliable prognostic biomarker (area under the curve: 0.757 [standard deviation 0.054, 95% confidence interval [CI] 0.650-0.864, p<0.001]) in progressive mCRPC, predicting PFS at 3 mo. Patients with LDH ≥450U/l were poorer PSA responders, with shorter PFS (213 vs 372 d, hazard ratio [HR] 1.876, 95% CI 1.289-2.7300) and OS (362 vs 563 d, HR 1.630, 95% CI 1.127-2.357). High LDH is an independent surrogate marker for survival following docetaxel and predicts a poor radiological response (p=0.043). Of the 14 patients with LDH ≥450U/l available for next-generation sequencing, nine (64.3%) were more likely to have DNA repair gene mutation(s) (BRCA1/2, ATM, CHEK2, Fanconi anaemia gene) in their primary biopsy. Cross correlation with MSKCC and SU2C/PCF databases revealed a positive correlation between LDHA, PARP1 (r=0.667, p<0.01), and other DNA repair genes. CONCLUSIONS: Genomic abnormalities of LDHA and DNA repair in primary biopsies link to high pretreatment LDH and poor response to docetaxel in mCRPC. PATIENT SUMMARY: The presence of mutations of the lactate dehydrogenase and DNA repair pathways are associated with aggressive prostate cancer and poor response to chemotherapy later in the disease.

Blocking Monocytic Myeloid-Derived Suppressor Cell Function via Anti-DC-HIL/GPNMB Antibody Restores the In Vitro Integrity of T Cells from Cancer Patients.

PURPOSE: Blocking the function of myeloid-derived suppressor cells (MDSC) is an attractive approach for cancer immunotherapy. Having shown DC-HIL/GPNMB to be the T-cell-inhibitory receptor mediating the suppressor function of MDSCs, we evaluated the potential of anti-DC-HIL mAb as an MDSC-targeting cancer treatment. EXPERIMENTAL DESIGN: Patients with metastatic cancer (n = 198) were analyzed by flow cytometry for DC-HIL or PDL1 expression on blood CD14+HLA-DRno/lo MDSCs. Their suppressor function was assessed by in vitro coculture with autologous T cells, and the ability of anti-DC-HIL or anti-PDL1 mAb to reverse such function was determined. Tumor expression of these receptors was examined histologically, and the antitumor activity of the mAb was evaluated by attenuated growth of colon cancers in mice. RESULTS: Patients with metastatic cancer had high blood levels of DC-HIL+ MDSCs compared with healthy controls. Anti-DC-HIL mAb reversed the in vitro function in ∼80% of cancer patients tested, particularly for colon cancer. Despite very low expression on blood MDSCs, anti-PDL1 mAb was as effective as anti-DC-HIL mAb in reversing MDSC function, a paradoxical phenomenon we found to be due to upregulated expression of PDL1 by T-cell-derived IFNγ in cocultures. DC-HIL is not expressed by colorectal cancer cells but by CD14+ cells infiltrating the tumor. Finally, anti-DC-HIL mAb attenuated growth of preestablished colon tumors by reducing MDSCs and increasing IFNγ-secreting T cells in the tumor microenvironment, with similar outcomes to anti-PDL1 mAb. CONCLUSIONS: Blocking DC-HIL function is a potentially useful treatment for at least colorectal cancer with high blood levels of DC-HIL+ MDSCs.See related commentary by Colombo, p. 453.

NF1 heterozygosity fosters de novo tumorigenesis but impairs malignant transformation.

Neurofibromatosis type 1 (NF1) is an autosomal genetic disorder. Patients with NF1 are associated with mono-allelic loss of the tumor suppressor gene NF1 in their germline, which predisposes them to develop a wide array of benign lesions. Intriguingly, recent sequencing efforts revealed that the NF1 gene is frequently mutated in multiple malignant tumors not typically associated with NF1 patients, suggesting that NF1 heterozygosity is refractory to at least some cancer types. In two orthogonal mouse models representing NF1- and non-NF1-related tumors, we discover that an Nf1+/- microenvironment accelerates the formation of benign tumors but impairs further progression to malignancy. Analysis of benign and malignant tumors commonly associated with NF1 patients, as well as those with high NF1 gene mutation frequency, reveals an antagonistic role for NF1 heterozygosity in tumor initiation and malignant transformation and helps to reconciliate the role of the NF1 gene in both NF1 and non-NF1 patient contexts.

Melanoma-Derived Soluble DC-HIL/GPNMB Promotes Metastasis by Excluding T-Lymphocytes from the Pre-Metastatic Niches.

Soluble factors from the primary tumor induce recruitment of bone marrow-derived progenitors to form tumor-supportive microenvironments or pre-metastatic niches in distal organs before metastasis. How tumor-secreted factors condition the sites for tumor progression remains ambiguous. B16 melanoma produces the secreted form of T cell-inhibitory DC-HIL (sDC-HIL) that travels to distal organs and potentiates the metastatic capacity of tumor cells. We studied the molecular mechanisms and found that sDC-HIL binds to select endothelial cells that co-localize with the sites where bone marrow-derived progenitors and tumor cells migrate. sDC-HIL-bound endothelial cells exist at a similar frequency in mice with or without tumors, and they are strongly associated with survival of intravenously injected tumor cells in the lung. sDC-HIL binding conferred T-cell suppressor function on the ECs and awakened the angiogenic property by inducing vascular endothelial growth factor expression, resulting in enhanced transendothelial migration of bone marrow-derived progenitors and tumor cells, but not for T cells. This selectivity is achieved by the T-cell binding of sDC-HIL, which prevents formation of the leading edges required for chemotaxis. Finally, inducing tumor expression of sDC-HIL significantly reduced tumor-infiltrated T cells. Therefore, the highly metastatic attribute of B16 melanoma can be explained by the endothelial gatekeeper function of sDC-HIL that limits lymphocyte transmigration to pre-metastatic niches.

Massively multiplex single-cell Hi-C.

We present single-cell combinatorial indexed Hi-C (sciHi-C), a method that applies combinatorial cellular indexing to chromosome conformation capture. In this proof of concept, we generate and sequence six sciHi-C libraries comprising a total of 10,696 single cells. We use sciHi-C data to separate cells by karyotypic and cell-cycle state differences and identify cell-to-cell heterogeneity in mammalian chromosomal conformation. Our results demonstrate that combinatorial indexing is a generalizable strategy for single-cell genomics.