Minimalist approaches to cancer tissue-of-origin classification by DNA methylation.

Classification of cancers by tissue-of-origin is fundamental to diagnostic pathology. While the combination of clinical data, tissue histology, and immunohistochemistry is usually sufficient, there remains a small but not insignificant proportion of difficult-to-classify cases. These challenging cases provide justification for ancillary molecular testing, including high-throughput DNA methylation array profiling, which promises cell-of-origin information and compatibility with formalin-fixed specimens. While diagnostically powerful, methylation profiling platforms are costly and technically challenging to implement, particularly for less well-resourced laboratories. To address this, we simulated the performance of "minimalist" methylation-based tests for cancer classification using publicly-available and internal institutional profiling data. These analyses showed that small and focused sets of the most informative CpG biomarkers from the arrays are sufficient for accurate diagnoses. As an illustrative example, one classifier, using information from just 53 out of about 450,000 available CpG probes, achieved an accuracy of 94.5% on 2575 fresh primary validation cases across 28 cancer types from The Cancer Genome Atlas Network. By training minimalist classifiers on formalin-fixed primary and metastatic cases, generally high accuracies were also achieved on additional datasets. These results support the potential of minimalist methylation testing, possibly via quantitative PCR and targeted next-generation sequencing platforms, in cancer classification.

Impact of multi-gene mutational profiling on clinical trial outcomes in metastatic breast cancer.

PURPOSE: Next-generation sequencing (NGS) has identified recurrent genomic alterations in metastatic breast cancer (MBC); however, the clinical utility of incorporating routine sequencing to guide treatment decisions in this setting is unclear. We examine the frequency of genomic alterations in MBC patients from academic and community hospitals and correlate with clinical outcomes. METHODS: MBC patients with good performance status were prospectively recruited at the Princess Margaret Cancer Centre (PM) in Canada. Molecular profiling on DNA extracted from FFPE archival tissues was performed on the Sequenom MassArray platform or the TruSeq Amplicon Cancer Panel (TSACP) on the MiSeq platform. Clinical trial outcomes by RECIST 1.1 and time on treatment were reviewed retrospectively. RESULTS: From January 2012 to November 2015, 483 MBC patients were enrolled and 440 were genotyped. At least one somatic mutation was identified in 46% of patients, most commonly in PIK3CA (28%) or TP53 (13%). Of 203 patients with ≥ 1 mutation(s), 15% were treated on genotype-matched and 9% on non-matched trials. There was no significant difference for median time on treatment for patients treated on matched vs. non-matched therapies (3.6 vs. 3.8 months; p = 0.89). CONCLUSIONS: This study provides real-world outcomes on hotspot genotyping and small targeted panel sequencing of MBC patients from academic and community settings. Few patients were matched to clinical trials with targeted therapies. More comprehensive profiling and improved access to clinical trials may increase therapeutic options for patients with actionable mutations. Further studies are needed to evaluate if this approach leads to improved clinical outcomes.

Clinical Utility of Tumor Next-Generation Sequencing Panel Testing to Inform Treatment Decisions for Patients With Advanced Solid Tumors in a Tertiary Care Center.

PURPOSE: There is limited information about the clinical utility of targeted next-generation sequencing (NGS) panel testing to inform decision making for patients with advanced solid tumors. The Ontario-wide Cancer Targeted Nucleic Acid Evaluation (OCTANE) is a prospective study that enrolled more than 4,500 patients with solid tumor for NGS panel testing. We performed a retrospective survey of medical oncologists to evaluate the impact of NGS testing on treatment decisions. METHODS: Patients and treating oncologists were identified at the Princess Margaret Cancer Center between 2016 and 2021. Tumor-only sequencing was performed using a gene panel of either 555 or 161 cancer genes. Oncologists were asked to review testing results and complete a survey indicating whether NGS testing affected treatment decisions. The primary outcome of this study was rate of treatment change on the basis of mutation results. Patient, test, and physician factors were evaluated for association with treatment changes using univariate analyses and a mixed-effects model. RESULTS: Of the 582 surveys sent, 394 (67.7%) were completed. We found that 188 (47.7%) patients had testing results classified as actionable by the oncologist and 62 (15.7%) patients were matched to treatment, of whom 37 (60%) were enrolled in a clinical trial, 13 (21%) received an approved drug, four (6%) were prescribed off-label therapy, and eight (13%) avoided ineffective treatment. Patient, test, and physician characteristics were not significantly associated with treatment change. There was no difference in overall survival between patients who received matched treatment versus those who did not (P = .55, median survival not reached). CONCLUSION: OCTANE testing led to a change in drug treatment in 15.7% of patients, supporting the clinical utility of NGS panel testing for patients with advanced solid tumors.

Impact on costs and outcomes of multi-gene panel testing for advanced solid malignancies: a cost-consequence analysis using linked administrative data.

Background: To date, economic analyses of tissue-based next generation sequencing genomic profiling (NGS) for advanced solid tumors have typically required models with assumptions, with little real-world evidence on overall survival (OS), clinical trial enrollment or end-of-life quality of care. Methods: Cost consequence analysis of NGS testing (555 or 161-gene panels) for advanced solid tumors through the OCTANE clinical trial (NCT02906943). This is a longitudinal, propensity score-matched retrospective cohort study in Ontario, Canada using linked administrative data. Patients enrolled in OCTANE at Princess Margaret Cancer Centre from August 2016 until March 2019 were matched with contemporary patients without large gene panel testing from across Ontario not enrolled in OCTANE. Patients were matched according to 19 patient, disease and treatment variables. Full 2-year follow-up data was available. Sensitivity analyses considered alternative matched cohorts. Main Outcomes were mean per capita costs (2019 Canadian dollars) from a public payer's perspective, OS, clinical trial enrollment and end-of-life quality metrics. Findings: There were 782 OCTANE patients with 782 matched controls. Variables were balanced after matching (standardized difference <0.10). There were higher mean health-care costs with OCTANE ($79,702 vs. $59,550), mainly due to outpatient and specialist visits. Publicly funded drug costs were less with OCTANE ($20,015 vs. $24,465). OCTANE enrollment was not associated with improved OS (restricted mean survival time [standard error]: 1.50 (±0.03) vs. 1.44 (±0.03) years, log-rank p = 0.153), varying by tumor type. In five tumor types with ≥35 OCTANE patients, OS was similar in three (breast, colon, uterus, all p > 0.40), and greater in two (ovary, biliary, both p < 0.05). OCTANE was associated with greater clinical trial enrollment (25.4% vs. 9.5%, p < 0.001) and better end-of-life quality due to less death in hospital (10.2% vs. 16.4%, p = 0.003). Results were robust in sensitivity analysis. Interpretation: We found an increase in healthcare costs associated with multi-gene panel testing for advanced cancer treatment. The impact on OS was not significant, but varied across tumor types. OCTANE was associated with greater trial enrollment, lower publicly funded drug costs and fewer in-hospital deaths suggesting important considerations in determining the value of NGS panel testing for advanced cancers. Funding: T.P H holds a research grant provided by the Ontario Institute for Cancer Research through funding provided by the Government of Ontario (#IA-035 and P.HSR.158) and through funding of the Canadian Network for Learning Healthcare Systems and Cost-Effective 'Omics Innovation (CLEO) via Genome Canada (G05CHS).

Genomic Characterization and Clinical Outcomes of Patients with Peritoneal Metastases from the AACR GENIE Biopharma Collaborative Colorectal Cancer Registry.

Peritoneal metastases (PM) are common in metastatic colorectal cancer (mCRC). We aimed to characterize patients with mCRC and PM from a clinical and molecular perspective using the American Association of Cancer Research Genomics Evidence Neoplasia Information Exchange (GENIE) Biopharma Collaborative (BPC) registry. Patients' tumor samples underwent targeted next-generation sequencing. Clinical characteristics and treatment outcomes were collected retrospectively. Overall survival (OS) from advanced disease and progression-free survival (PFS) from start of cancer-directed drug regimen were estimated and adjusted for the left truncation bias. A total of 1,281 patients were analyzed, 244 (19%) had PM at time of advanced disease. PM were associated with female sex [OR: 1.67; 95% confidence interval (CI): 1.11-2.54; P = 0.014] and higher histologic grade (OR: 1.72; 95% CI: 1.08-2.71; P = 0.022), while rectal primary tumors were less frequent in patients with PM (OR: 0.51; 95% CI: 0.29-0.88; P < 0.001). APC occurred less frequently in patients with PM (N = 151, 64% vs. N = 788, 79%) while MED12 alterations occurred more frequently in patients with PM (N = 20, 10% vs. N = 32, 4%); differences in MED12 were not significant when restricting to oncogenic and likely oncogenic variants according to OncoKB. Patients with PM had worse OS (HR: 1.45; 95% CI: 1.16-1.81) after adjustment for independently significant clinical and genomic predictors. PFS from initiation of first-line treatment did not differ by presence of PM. In conclusion, PM were more frequent in females and right-sided primary tumors. Differences in frequencies of MED12 and APC alterations were identified between patients with and without PM. PM were associated with shorter OS but not with PFS from first-line treatment. SIGNIFICANCE: Utilizing the GENIE BPC registry, this study found that PM in patients with colorectal cancer occur more frequently in females and right-sided primary tumors and are associated with worse OS. In addition, we found a lower frequency of APC alterations and a higher frequency in MED12 alterations in patients with PM.

The GENIE BPC NSCLC Cohort: A Real-World Repository Integrating Standardized Clinical and Genomic Data for 1,846 Patients with Non-Small Cell Lung Cancer.

PURPOSE: We describe the clinical and genomic landscape of the non-small cell lung cancer (NSCLC) cohort of the American Association for Cancer Research (AACR) Project Genomics Evidence Neoplasia Information Exchange (GENIE) Biopharma Collaborative (BPC). EXPERIMENTAL DESIGN: A total of 1,846 patients with NSCLC whose tumors were sequenced from 2014 to 2018 at four institutions participating in AACR GENIE were randomly chosen for curation using the PRISSMM data model. Progression-free survival (PFS) and overall survival (OS) were estimated for patients treated with standard therapies. RESULTS: In this cohort, 44% of tumors harbored a targetable oncogenic alteration, with EGFR (20%), KRAS G12C (13%), and oncogenic fusions (ALK, RET, and ROS1; 5%) as the most frequent. Median OS (mOS) on first-line platinum-based therapy without immunotherapy was 17.4 months [95% confidence interval (CI), 14.9-19.5 months]. For second-line therapies, mOS was 9.2 months (95% CI, 7.5-11.3 months) for immune checkpoint inhibitors (ICI) and 6.4 months (95% CI, 5.1-8.1 months) for docetaxel ± ramucirumab. In a subset of patients treated with ICI in the second-line or later setting, median RECIST PFS (2.5 months; 95% CI, 2.2-2.8) and median real-world PFS based on imaging reports (2.2 months; 95% CI, 1.7-2.6) were similar. In exploratory analysis of the impact of tumor mutational burden (TMB) on survival on ICI treatment in the second-line or higher setting, TMB z-score harmonized across gene panels was associated with improved OS (univariable HR, 0.85; P = 0.03; n = 247 patients). CONCLUSIONS: The GENIE BPC cohort provides comprehensive clinicogenomic data for patients with NSCLC, which can improve understanding of real-world patient outcomes.

A Scalable Quality Assurance Process for Curating Oncology Electronic Health Records: The Project GENIE Biopharma Collaborative Approach.

PURPOSE: The American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange Biopharma Collaborative is a multi-institution effort to build a pan-cancer repository of genomic and clinical data curated from the electronic health record. For the research community to be confident that data extracted from electronic health record text are reliable, transparency of the approach used to ensure data quality is essential. MATERIALS AND METHODS: Four institutions participating in AACR's Project GENIE created an observational cohort of patients with cancer for whom tumor molecular profiling data, therapeutic exposures, and treatment outcomes are available and will be shared publicly with the research community. A comprehensive approach to quality assurance included assessments of (1) feasibility of the curation model through pressure test cases; (2) accuracy through programmatic queries and comparison with source data; and (3) reproducibility via double curation and code review. RESULTS: Assessments of feasibility resulted in critical modifications to the curation directives. Queries and comparison with source data identified errors that were rectified via data correction and curator retraining. Assessment of intercurator reliability indicated a reliable curation model. CONCLUSION: The transparent quality assurance processes for the GENIE BPC data ensure that the data can be used for analyses that support clinical decision making and advances in precision oncology.

Natural History and Characteristics of ERBB2-mutated Hormone Receptor-positive Metastatic Breast Cancer: A Multi-institutional Retrospective Case-control Study from AACR Project GENIE.

PURPOSE: We wanted to determine the prognosis and the phenotypic characteristics of hormone receptor-positive advanced breast cancer tumors harboring an ERBB2 mutation in the absence of a HER2 amplification. EXPERIMENTAL DESIGN: We retrospectively collected information from the American Association of Cancer Research-Genomics Evidence Neoplasia Information Exchange registry database from patients with hormone receptor-positive, HER2-negative, ERBB2-mutated advanced breast cancer. Phenotypic and co-mutational features, as well as response to treatment and outcome were compared with matched control cases ERBB2 wild type. RESULTS: A total of 45 ERBB2-mutant cases were identified for 90 matched controls. The presence of an ERBB2 mutation was not associated with worse outcome determined by overall survival (OS) from first metastatic relapse. No significant differences were observed in phenotypic characteristics apart from higher lobular infiltrating subtype in the ERBB2-mutated group. ERBB2 mutation did not seem to have an impact in response to treatment or time-to-progression (TTP) to endocrine therapy compared with ERBB2 wild type. In the co-mutational analyses, CDH1 mutation was more frequent in the ERBB2-mutated group (FDR < 1). Although not significant, fewer co-occurring ESR1 mutations and more KRAS mutations were identified in the ERBB2-mutated group. CONCLUSIONS: ERBB2-activating mutation was not associated with a worse OS from time of first metastatic relapse, or differences in TTP on treatment as compared with a series of matched controls. Although not significant, differences in coexisting mutations (CDH1, ESR1, and KRAS) were noted between the ERBB2-mutated and the control group.

Correlation Between Surrogate End Points and Overall Survival in a Multi-institutional Clinicogenomic Cohort of Patients With Non-Small Cell Lung or Colorectal Cancer.

Importance: Contemporary observational cancer research requires associating genomic biomarkers with reproducible end points; overall survival (OS) is a key end point, but interpretation can be challenging when multiple lines of therapy and prolonged survival are common. Progression-free survival (PFS), time to treatment discontinuation (TTD), and time to next treatment (TTNT) are alternative end points, but their utility as surrogates for OS in real-world clinicogenomic data sets has not been well characterized. Objective: To measure correlations between candidate surrogate end points and OS in a multi-institutional clinicogenomic data set. Design, Setting, and Participants: A retrospective cohort study was conducted of patients with non-small cell lung cancer (NSCLC) or colorectal cancer (CRC) whose tumors were genotyped at 4 academic centers from January 1, 2014, to December 31, 2017, and who initiated systemic therapy for advanced disease. Patients were followed up through August 31, 2020 (NSCLC), and October 31, 2020 (CRC). Statistical analyses were conducted on January 5, 2021. Exposures: Candidate surrogate end points included TTD; TTNT; PFS based on imaging reports only; PFS based on medical oncologist ascertainment only; PFS based on either imaging or medical oncologist ascertainment, whichever came first; and PFS defined by a requirement that both imaging and medical oncologist ascertainment have indicated progression. Main Outcomes and Measures: The primary outcome was the correlation between candidate surrogate end points and OS. Results: There were 1161 patients with NSCLC (648 women [55.8%]; mean [SD] age, 63 [11] years) and 1150 with CRC (647 men [56.3%]; mean [SD] age, 54 [12] years) identified for analysis. Progression-free survival based on both imaging and medical oncologist documentation was most correlated with OS (NSCLC: ρ = 0.76; 95% CI, 0.73-0.79; CRC: ρ = 0.73; 95% CI, 0.69-0.75). Time to treatment discontinuation was least associated with OS (NSCLC: ρ = 0.45; 95% CI, 0.40-0.50; CRC: ρ = 0.13; 95% CI, 0.06-0.19). Time to next treatment was modestly associated with OS (NSCLC: ρ = 0.60; 0.55-0.64; CRC: ρ = 0.39; 95% CI, 0.32-0.46). Conclusions and Relevance: This cohort study suggests that PFS based on both a radiologist and a treating oncologist determining that a progression event has occurred was the surrogate end point most highly correlated with OS for analysis of observational clinicogenomic data.

Latency and interval therapy affect the evolution in metastatic colorectal cancer.

While comparison of primary tumor and metastases has highlighted genomic heterogeneity in colorectal cancer (CRC), previous studies have focused on a single metastatic site or limited genomic testing. Combining data from whole exome and ultra-deep targeted sequencing, we explored possible evolutionary trajectories beyond the status of these mutations, particularly among patient-matched metastatic tumors. Our findings confirm the persistence of known clinically-relevant mutations (e.g., those of RAS family of oncogenes) in CRC primary and metastases, yet reveal that latency and interval systemic therapy affect the course of evolutionary events within metastatic lesions. Specifically, our analysis of patient-matched primary and multiple metastatic lesions, developed over time, showed a similar genetic composition for liver metastatic tumors, which were 21-months apart. This genetic makeup was different from those identified in lung metastases developed before manifestation of the second liver metastasis. These results underscore the role of latency in the evolutionary path of metastatic CRC and may have implications for future treatment options.

Characteristics and Outcome of AKT1 E17K-Mutant Breast Cancer Defined through AACR Project GENIE, a Clinicogenomic Registry.

AKT inhibitors have promising activity in AKT1 E17K-mutant estrogen receptor (ER)-positive metastatic breast cancer, but the natural history of this rare genomic subtype remains unknown. Utilizing AACR Project GENIE, an international clinicogenomic data-sharing consortium, we conducted a comparative analysis of clinical outcomes of patients with matched AKT1 E17K-mutant (n = 153) and AKT1-wild-type (n = 302) metastatic breast cancer. AKT1-mutant cases had similar adjusted overall survival (OS) compared with AKT1-wild-type controls (median OS, 24.1 vs. 29.9, respectively; P = 0.98). AKT1-mutant cases enjoyed longer durations on mTOR inhibitor therapy, an observation previously unrecognized in pivotal clinical trials due to the rarity of this alteration. Other baseline clinicopathologic features, as well as durations on other classes of therapy, were broadly similar. In summary, we demonstrate the feasibility of using a novel and publicly accessible clincogenomic registry to define outcomes in a rare genomically defined cancer subtype, an approach with broad applicability to precision oncology. SIGNIFICANCE: We delineate the natural history of a rare genomically distinct cancer, AKT1 E17K-mutant ER-positive breast cancer, using a publicly accessible registry of real-world patient data, thereby illustrating the potential to inform drug registration through synthetic control data.See related commentary by Castellanos and Baxi, p. 490.

Molecular Profiling of Patients With Advanced Colorectal Cancer: Princess Margaret Cancer Centre Experience.

BACKGROUND: Molecular aberrations in KRAS, NRAS, BRAF, and PIK3CA have been well-described in advanced colorectal cancer. The incidences of other mutations are less known. We report results of molecular profiling of advanced colorectal cancer in an academic cancer center. PATIENTS AND METHODS: Patients with advanced colorectal were enrolled in an institution-wide molecular profiling program. Profiling was performed on formalin-fixed paraffin embedded archival tissues using a customized MassArray panel (23 genes, 279 mutations) or the Illumina MiSeq TruSeq Cancer Panel (48 genes, 212 amplicons, ≥ 500× coverage) in a Clinical Laboratory Improvement Amendments-certified laboratory. PTEN was determined by immunohistochemistry. RESULTS: From March 2012 to April 2014, 245 patients were enrolled. At least one mutation was found in 54% (97/178) and 91% (61/67) of patients using MassArray or MiSeq platforms, respectively (P < .01). Of all patients, KRAS G12/13 mutation was identified in 39%, and non-G12/13 KRAS, BRAF, or NRAS mutations were present in 9%, 6%, and 4%, respectively. Other common mutations included TP53 (68.7%), APC (41.8%), and PIK3CA (13.5%). Co-mutation with KRAS, NRAS, or BRAF was found in 75% of patients with PIK3CA mutation. Of 106 patients with known PTEN immunohistochemistry status, 16% were negative. A higher average number of mutations were observed in right versus left colorectal cancer (P < .01), with 13 of 14 BRAF mutations located in right colon cancer. CONCLUSION: Mutations are common in advanced colorectal cancer. Right colon cancers harbor more genetic aberrations than left colon or rectal cancers. These aberrations may contribute to differential outcomes to anti-epidermal growth factor receptor therapy among patients with right colon, left colon, or rectal cancers.

Minimally Invasive Real-Time Detection of Actionable Mutations in Patients With Metastatic Solid Tumors Using Fine-Needle and Liquid Biopsies.

PURPOSE: Fine-needle biopsy (FNB) and liquid biopsy are minimally invasive methods of tumor sampling that provide feasible means to assess tumor genotypes in real time. However, more data are needed to establish the strength of these methods by benchmarking against the current gold standard methods, core-needle biopsy (CNB) or surgical excision of the tumor. PATIENTS AND METHODS: Eligible patients with advanced solid tumors were prospectively recruited. We performed mutation profiling using matched tumor DNA obtained by CNB, FNB and liquid biopsy, and matrix-assisted laser desorption/ionization time-of-flight custom mass-spectrometry or targeted next-generation DNA sequencing. The actionability of detected mutations was determined using the OncoKB Web tool. Agreement between mutations detected in CNBs, FNBs, and circulating tumor DNA (ctDNA) was examined. RESULTS: Forty-one patients underwent tumor biopsy. Thirty CNBs (73%) and 34 FNBs (83%) had sufficient tumor and DNA for mutation profiling. Median DNA yield from CNB and FNB were 775 ng (interquartile range, 240 to 347 4ng) and 649 ng (interquartile range, 180 to1350 ng), respectively. Of 29 CNB/FNB pairs available for comparison, actionable mutation results were concordant in 28 (96%). Six of nine actionable mutations (67%) that were found by CNB, FNB, or both were detectable in ctDNA. Two additional actionable mutations were found exclusively in ctDNA. CONCLUSION: Optimally processed FNB and liquid biopsy can be used routinely for tumor mutation profiling to identify actionable mutations.

Molecular profiling of advanced solid tumors and patient outcomes with genotype-matched clinical trials: the Princess Margaret IMPACT/COMPACT trial.

BACKGROUND: The clinical utility of molecular profiling of tumor tissue to guide treatment of patients with advanced solid tumors is unknown. Our objectives were to evaluate the frequency of genomic alterations, clinical "actionability" of somatic variants, enrollment in mutation-targeted or other clinical trials, and outcome of molecular profiling for advanced solid tumor patients at the Princess Margaret Cancer Centre (PM). METHODS: Patients with advanced solid tumors aged ≥18 years, good performance status, and archival tumor tissue available were prospectively consented. DNA from archival formalin-fixed paraffin-embedded tumor tissue was tested using a MALDI-TOF MS hotspot panel or a targeted next generation sequencing (NGS) panel. Somatic variants were classified according to clinical actionability and an annotated report included in the electronic medical record. Oncologists were provided with summary tables of their patients' molecular profiling results and available mutation-specific clinical trials. Enrolment in genotype-matched versus genotype-unmatched clinical trials following release of profiling results and response by RECIST v1.1 criteria were evaluated. RESULTS: From March 2012 to July 2014, 1893 patients were enrolled and 1640 tested. After a median follow-up of 18 months, 245 patients (15 %) who were tested were subsequently treated on 277 therapeutic clinical trials, including 84 patients (5 %) on 89 genotype-matched trials. The overall response rate was higher in patients treated on genotype-matched trials (19 %) compared with genotype-unmatched trials (9 %; p < 0.026). In a multi-variable model, trial matching by genotype (p = 0.021) and female gender (p = 0.034) were the only factors associated with increased likelihood of treatment response. CONCLUSIONS: Few advanced solid tumor patients enrolled in a prospective institutional molecular profiling trial were treated subsequently on genotype-matched therapeutic trials. In this non-randomized comparison, genotype-enrichment of early phase clinical trials was associated with an increased objective tumor response rate. TRIAL REGISTRATION: NCT01505400 (date of registration 4 January 2012).

Expression and function of myometrial PSF suggest a role in progesterone withdrawal and the initiation of labor.

Progesterone (P4), acting through its receptor (PR), is essential for the maintenance of pregnancy. P4 acts by suppressing uterine contractility and the expression of contraction-associated proteins (CAP) such as connexin 43 (Cx43). P4 levels must be reduced or its actions blocked to allow the increased expression of CAP genes and the initiation of labor. Although the importance of progesterone in pregnancy has been known for about 80 yr, the fundamental mechanisms by which P4/PR maintains myometrial quiescence and by which this signaling is blocked at term labor remain to be determined. In this manuscript, we demonstrate that ligand-bound PR interacts with the Cx43 gene promoter through activator protein-1 transcription factors. We show that the ability of PR to repress Cx43 transcription is conferred through the recruitment of the PR coregulator, polypyrimidine tract binding protein-associated splicing factor (PSF), and the further recruitment of the yeast switch independent 3 homolog A/histone deacetylase corepressor complex. PSF expression is elevated during pregnancy but falls toward term as a result of increased mechanical stretch of the myometrium and a rise in the concentrations of circulating estrogen. These data together indicate that PSF is a critical regulator of P4/PR signaling and labor. We suggest that decreased PSF at term may result in a de-repression of PR transcriptional control of CAP genes and thereby contributes to a functional withdrawal of progesterone at term labor.

p54nrb is a transcriptional corepressor of the progesterone receptor that modulates transcription of the labor-associated gene, connexin 43 (Gja1).

The progesterone receptor (PR) plays important roles in the establishment and maintenance of pregnancy. By dynamic interactions with coregulators, PR represses the expression of genes that increase the contractile activity of myometrium and contribute to the initiation of labor. We have previously shown that PTB-associated RNA splicing factor (PSF) can function as a PR corepressor. In this report, we demonstrated that the PSF heterodimer partner, p54nrb (non-POU-domain-containing, octamer binding protein), can also function as a transcription corepressor, independent of PSF. p54nrb Interacts directly with PR independent of progesterone. In contrast to PSF, p54nrb neither enhances PR protein degradation nor blocks PR binding to DNA. Rather, p54nrb recruits mSin3A through its N terminus to the PR-DNA complex, resulting in an inhibition of PR-mediated transactivation of the progesterone-response element-luciferase reporter gene. PR also repressed transcription of the connexin 43 gene (Gja1), an effect dependent on the presence of an activator protein 1 site within the proximal Gja1 promoter. Mutation of this site abolished PR-mediated repression and decreased the recruitment of PR and p54nrb onto the Gja1 promoter. Furthermore, knockdown p54nrb expression by small interfering RNA alleviated PR-mediated repression on Gja1 transcription, whereas overexpression of p54nrb enhanced it. In the physiological context of pregnancy, p54nrb protein levels decrease with the approach of labor in the rat myometrium. We conclude that p54nrb is a transcriptional corepressor of PR. Decreased expression of p54nrb at the time of labor may act to derepress PR-mediated inhibition on connexin 43 expression and contribute to the initiation of labor.

Vaccinia virus activation of CCR5 invokes tyrosine phosphorylation signaling events that support virus replication.

Vaccinia virus, a poxvirus, produces structurally distinct forms of virions for which the immediate events following cell entry are ill-defined. We provide evidence that intracellular mature virus (IMV) enters both permissive and nonpermissive T-cell lines and that introduction of CCR5 into nonpermissive mouse fibroblasts or human primary T cells renders the cells permissive for vaccinia replication. Notably, T cells expressing CCR5 in which tyrosine 339 in the intracellular region is replaced by phenylalanine no longer support virus replication or virus-inducible activation of specific host cell signaling effectors IRS-2, Grb2, and Erk1/2. We show that following IMV entry into the cell, the intact but not the tyrosine-deficient CCR5 is rapidly internalized and colocalizes with virus. This colocalization precedes virus-inducible signaling and replication.