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In vivo anti-Gr1 Ab treatment fails to deplete CD11b+/Gr1+ myeloid cells in bone marrow of mice with skeletal tumors

Osteoclast (OC) blockade has been successful in reducing tumor growth in bone in preclinical settings, but antiresorptive drugs, such as zoledronic acid (ZA), fail to improve the overall survival rate of patients with bone metastasis despite ameliorating skeletal complications. To address this unmet clinical need, we interrogated what other cells modulated tumor growth in bone in addition to OCs. Because myeloid-derived suppressor cells (MDSC)—heterogeneous populations expressing CD11b, Ly6C, and Ly6G markers—originate in the bone marrow and promote tumor progression, we hypothesized that their accumulation hinders ZA antitumor effects. By using a murine model of bone metastasis insensitive to OC blockade, we assessed the antitumor effect of MDSC depletion using anti-Gr1 in mice bearing skeletal lung [Lewis lung carcinoma (LLC)], melanoma (B16-F10), and mammary (4T1) tumors. Differently from soft tissue tumors, anti-Gr1 did not reduce bone metastases and led to the paradoxical accumulation of bone marrow–resident CD11b+Ly6CintLy6Gint cells that differentiated into OCs when cultured in vitro. Anti-Gr1–mediated depletion of Ly6G+ granulocytic MDSCs combined with ZA-induced OC blockade reduced growth of established skeletal metastases compared with each agent alone. CD15+ granulocytic populations were increased in patients with breast cancer with progressive bone disease after antiresorptive treatment compared with those with stable bone disease. We provide evidence that antiresorptive therapies fail to reduce bone metastases in the presence of elevated granulocytic populations and that effective treatment of established skeletal metastases requires combinatorial depletion of granulocytes and OC blockade.

46 Background: The KEYNOTE-177 randomized controlled trial demonstrated that mCRC patients with MSI high (MSI-H) and/or deficient MMR (dMMR) have better outcomes on first-line ICI than chemotherapy. This study aimed to evaluate MSI by next-generation sequencing (NGS) as a predictive biomarker of ICI effectiveness in real-world settings, and compare MSI (NGS), dMMR (immunohistochemistry, IHC), and TMB (10 mut/Mb cutoff) in predicting ICI outcomes. Methods: A prospectively declared statistical analysis plan compared the effectiveness of first-line treatment ICI versus chemotherapy in patients with MSI-H mCRC, and compared the predictive power of ICI outcomes associated with biomarkers in any line of therapy (LOT). Patient data was obtained by the US-based de-identified Flatiron Health-Foundation Medicine real-world mCRC clinico-genomic database (FH-FMI CGDB), originating from ~280 US cancer clinics between Jan/2011 and Dec/2021. Differences in progression-free survival (PFS) and overall survival (OS) were evaluated by Cox proportional hazard models, adjusted for a risk score generated from prognostic clinical features. The likelihood ratio test evaluated the superiority of a biomarker in anticipating outcomes. Results: Patients with MSI-H mCRC had more favorable outcomes receiving first-line ICI (n=49) than chemotherapy (n=89) for PFS (median 24.87 vs. 5.65 months, hazard ratio (HR): 0.31, 95% confidence interval (CI) 0.18-0.52, p 10, three with mutations in MMR genes, and 5 with BRAF mutation, consistent with MSI biology). When MSI (NGS) is added to dMMR (IHC) evaluation, there is an improvement in the prediction of TTNT (p = 0.0013), PFS (p = 0.0202), and OS (p = 0.0717), but adding MMR status to MSI did not improve explanatory power. Conclusions: MSI (NGS) robustly identifies mCRC patients with favorable outcomes on first-line ICI vs. chemotherapy, and is a nominally better predictor of ICI outcomes when compared with dMMR (IHC) alone. dMMR (IHC), MSI (NGS), and TMB have similar predictive power for ICI benefit. [Table: see text]

ImportanceThe KEYNOTE-177 trial demonstrated that patients with metastatic colorectal cancer (MCRC) with high microsatellite instability (MSI-H) and/or mismatch repair deficiency (DMMR) have better outcomes when receiving first-line immune checkpoint inhibitors (ICIs) compared with chemotherapy. Data on performance of ICIs in patients with MCRC in standard practice settings remain limited, and direct MMR vs MSI outcome association comparisons are lacking.ObjectiveTo validate MSI (determined by next-generation sequencing [NGS]) as a biomarker of ICI effectiveness among patients with MCRC in standard practice settings and examine the association of MSI assessed by NGS, DMMR by immunohistochemistry, and tumor mutational burden (cutoff, 10 mutations/megabase) with ICI outcomes.Design, Setting, and ParticipantsThis comparative effectiveness research study of outcomes in prospectively defined biomarker subgroups used data from a deidentified clinicogenomic database and included patients who received Foundation Medicine testing (FoundationOne or FoundationOne CDx) during routine clinical care at approximately 280 US academic or community-based cancer clinics between March 2014 and December 2021. The population included 1 cohort of patients with MSI-H MCRC who received first-line ICIs or chemotherapy and a second cohort who received ICIs in any line of therapy (LOT) for biomarker examination.ExposuresICI therapy or chemotherapy assigned at physician discretion without randomization.Main Outcomes and MeasuresThe main outcomes were time to next treatment (TTNT), progression-free survival (PFS), and overall survival (OS). Hazard ratios were adjusted for known prognostic imbalances. Comparisons of explanatory power used the likelihood ratio test.ResultsA total of 138 patients (median age, 67.0 years [IQR, 56.2-74.0 years]; 73 [52.9%] female) with MSI-H MCRC received first-line ICIs or chemotherapy. A total of 182 patients (median age, 64.5 years [IQR, 55.2-72.0]; 98 [53.8%] female) received ICIs in any LOT. Patients receiving first-line ICIs vs chemotherapy had longer TTNT (median, not reached [NR] vs 7.23 months [IQR, 6.21-9.72 months]; adjusted hazard ratio [AHR], 0.17; 95% CI, 0.08-0.35; P P P = .02). MSI added to DMMR better anticipated TTNT and PFS in patients receiving ICIs than DMMR alone. The same was not observed when DMMR evaluation was added to MSI.Conclusions and RelevanceIn this comparative effectiveness research study, MSI assessed by NGS robustly identified patients with favorable outcomes on first-line ICIs vs chemotherapy and appeared to better anticipate ICI outcomes compared with DMMR.

The aggressive biology of pancreatic ductal adenocarcinoma (PDAC), along with its limited sensitivity to many systemic therapies, presents a major challenge in the management of patients with metastatic PDAC. Over the past decade, the incorporation of combinatorial cytotoxic chemotherapy regimens has improved patient outcomes. Despite these advances, resistance to cytotoxic chemotherapy inevitably occurs, and there is a great need for effective therapies. A major focus of research has been to identify molecularly defined subpopulations of patients with PDAC who may benefit from targeted therapies that are matched to their molecular profile. Recent successes include the demonstration of the efficacy of maintenance PARP inhibition in PDAC tumors harboring deleterious BRCA1, BRCA2, and PALB2 alterations. In addition, while therapeutic targeting of KRAS was long thought to be infeasible, emerging data on the efficacy of KRAS G12C inhibitors have increased optimism about next-generation KRAS-directed therapies in PDAC. Meanwhile, KRAS wild-type PDAC encompasses a unique molecular subpopulation of PDAC that is enriched for targetable genetic alterations, such as oncogenic BRAF alterations, mismatch repair deficiency, and FGFR2, ALK, NTRK, ROS1, NRG1, and RET rearrangements. As more molecularly targeted therapies are developed, precision medicine has the potential to revolutionize the treatment of patients with metastatic PDAC.

Osteoclast (OC) blockade has been successful in reducing tumor growth in bone in preclinical settings, but antiresorptive drugs, such as zoledronic acid (ZA), fail to improve the overall survival rate of patients with bone metastasis despite ameliorating skeletal complications. To address this unmet clinical need, we interrogated what other cells modulated tumor growth in bone in addition to OCs. Because myeloid-derived suppressor cells (MDSC)—heterogeneous populations expressing CD11b, Ly6C, and Ly6G markers—originate in the bone marrow and promote tumor progression, we hypothesized that their accumulation hinders ZA antitumor effects. By using a murine model of bone metastasis insensitive to OC blockade, we assessed the antitumor effect of MDSC depletion using anti-Gr1 in mice bearing skeletal lung [Lewis lung carcinoma (LLC)], melanoma (B16-F10), and mammary (4T1) tumors. Differently from soft tissue tumors, anti-Gr1 did not reduce bone metastases and led to the paradoxical accumulation of bone marrow–resident CD11b+Ly6CintLy6Gint cells that differentiated into OCs when cultured in vitro. Anti-Gr1–mediated depletion of Ly6G+ granulocytic MDSCs combined with ZA-induced OC blockade reduced growth of established skeletal metastases compared with each agent alone. CD15+ granulocytic populations were increased in patients with breast cancer with progressive bone disease after antiresorptive treatment compared with those with stable bone disease. We provide evidence that antiresorptive therapies fail to reduce bone metastases in the presence of elevated granulocytic populations and that effective treatment of established skeletal metastases requires combinatorial depletion of granulocytes and OC blockade.

PURPOSE Cell-free DNA (cfDNA) and circulating tumor cell (CTC)–based liquid biopsies have emerged as potential tools to predict responses to androgen receptor (AR)–directed therapy in metastatic prostate cancer. However, because of complex mechanisms and incomplete understanding of genomic events involved in metastatic prostate cancer resistance, current assays (eg, CTC AR-V7) demonstrate low sensitivity and remain underutilized. The recent discovery of AR enhancer amplification in > 80% of patients with metastatic disease and its association with disease resistance presents an opportunity to improve on current assays. We hypothesized that tracking AR/enhancer genomic alterations in plasma cfDNA would detect resistance with high sensitivity and specificity. PATIENTS AND METHODS We developed a targeted sequencing and analysis method as part of a new assay called Enhancer and Neighboring Loci of Androgen Receptor Sequencing (EnhanceAR-Seq). We applied EnhanceAR-Seq to plasma collected from 40 patients with metastatic prostate cancer treated with AR-directed therapy to monitor AR/enhancer genomic alterations and correlated these events with therapy resistance, progression-free survival (PFS), and overall survival (OS). RESULTS EnhanceAR-Seq identified genomic alterations in the AR/enhancer locus in 45% of cases, including a 40% rate of AR enhancer amplification. Patients with AR/enhancer alterations had significantly worse PFS and OS than those without (6-month PFS, 30% v 71%; P = .0002; 6-month OS, 59% v 100%; P = .0015). AR/enhancer alterations in plasma cfDNA detected 18 of 23 resistant cases (78%) and outperformed the CTC AR-V7 assay, which was also run on a subset of patients. CONCLUSION cfDNA-based AR locus alterations, including of the enhancer, are strongly associated with resistance to AR-directed therapy and significantly worse survival. cfDNA analysis using EnhanceAR-Seq may enable more precise risk stratification and personalized therapeutic approaches for metastatic prostate cancer.

The phosphatidylinositol 3-kinase (PI3K) pathway is the most common aberrantly activated pathway in breast cancer, making it an attractive therapeutic target. In this review, we will discuss the rationale for targeting PI3K/AKT signaling and the development of PI3K/AKT inhibitors in breast cancer. Although the initial clinical trials with pan-PI3K inhibitors were challenged by high toxicities and modest antitumor effect, there has been continued effort to develop agents more precisely targeting PI3K isoforms to improve therapeutic index. Alpelisib in combination with fulvestrant is now available in the clinic for postmenopausal women with advanced or metastatic hormone receptor (HR)-positive, HER2-negative, PIK3CA-mutated breast cancer. In addition, promising data has been observed in randomized phase II trials of AKT inhibitors in combination with fulvestrant or paclitaxel in metastatic HR-positive, HER2-negative disease and triple negative breast cancer (TNBC), respectively. The high frequency of genetic alterations in the PI3K pathway has provided the rationale for development of inhibitors targeting PI3K/AKT. Despite initial disappointment with several randomized trials of pan-PI3K inhibitors in HR-positive breast cancer, there has been continued effort to more precisely target PI3K isoforms, which has led to clinical benefit for patients with advanced breast cancer.

Introduction: Analysis of cell-free DNA (cfDNA) has recently emerged as a non-invasive modality for guiding cancer diagnostics and treatment decisions. However, efforts have predominantly focused on the analysis of single nucleotide variants (SNVs) and insertions/deletions (indels). Despite the clinical significance of many larger variants such as structural variations (SVs) and copy number alterations (CNAs), detecting them in cfDNA remains a challenge. Given the lack of existing tools we seek to develop an integrated bioinformatic pipeline for SV and CNA detection in cfDNA following targeted hybrid-capture next-generation sequencing (NGS), along with standard SNV and indel analysis. Methods: SVs were first detected using Manta, Lumpy and Delly in plasma cfDNA in comparison with matched peripheral blood leukocyte (PBL) DNA samples from cancer patients, then combined to identify consensus SVs and genotyped throughout samples from patients and healthy individuals. Next, consensus SVs were called somatic events if they were supported by split reads and discordant read pairs in cfDNA samples from patients but not in matched PBL or healthy donor cfDNA samples. For CNA analysis, the ratio of read depth between patient-derived plasma cfDNA and a panel of healthy controls was calculated across genomic bins using the CNVkit tool, followed by bias correction and recentralization using CNA negative control genes to account for read coverage imbalances in targeted NGS. Last, SNV and indel analysis was integrated from the CAPP-Seq pipeline. Results: We applied our pipeline to targeted hybrid-capture NGS data from 48 patients across two independent cohorts of metastatic castration resistant prostate cancer (mCRPC). The targeted panel covered the full-length AR gene body and a hotspot region of TMPRSS2-ERG fusion break points. Consistent with earlier whole genome studies, we confirmed known CNAs and SVs in tumor suppressors, oncogenes and regulatory elements including AR gene and AR enhancer duplications (22/48, 46% of patients), TMPRSS2-ERG gene fusions (9/48, 19%), PTEN and TP53 loss (8/48, 17%). Notably, our pipeline outperformed FACTERA which did not detect any TMPRSS2-ERG gene fusions or AR/enhancer tandem duplications. Subsequent analysis showed high concordance between plasma cfDNA and matched tumor biopsies, and our pipeline recapitulated the landscape of SVs and CNAs in an in silico cfDNA simulation from tumor biopsies. Finally, we showed that alterations of the AR/enhancer locus detected by our pipeline were strongly associated with treatment resistance, patient progression-free and overall survival in mCRPC. Conclusion: We developed a unified pipeline for detection of SVs, CNAs and small mutations in cfDNA targeted sequencing with potential applications in monitoring cancer progression and predicting patient treatment response. Citation Format: Ha X. Dang, Jace Webster, Pradeep S. Chauhan, Steven H. Hartman, Wenjia Feng, Elisa M. Ledet, Haley Ellis, Patrick J. Miller, Ellen B. Jaeger, Sydney A. Caputo, Peter K. Harris, A. Oliver Sartor, Russell K. Pachynski, Aadel A. Chaudhuri, Christopher A. Maher. A unified pipeline to detect small mutations, structural variations and copy number alterations from targeted cell-free DNA sequencing in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 577.

Background: We previously developed a liquid biopsy assay called Enhancer and neighboring loci of Androgen Receptor Sequencing (EnhanceAR-Seq) (Dang & Chauhan et al, JCO PO, 2020). We applied it to a heterogeneous cohort of metastatic prostate cancer patients after the start of AR-directed therapy, and showed that alterations in the AR locus were associated with worse survival. Here we asked if AR/enhancer genomic alterations detected in plasma cell-free DNA prior to the administration of first-line AR-selective inhibitors (ARSIs) can predict survival in metastatic castration resistant prostate cancer (mCRPC) patients. Methods: We applied EnhanceAR-Seq to plasma cell-free DNA isolated from 20 mCRPC patients from Tulane University collected between April 2015 and June 2017. Assay results were correlated with patient overall survival (OS) and progression-free survival (PFS) from the time of blood collection. Results: Median follow up time was 32 months. Seventeen patients had blood plasma analyzed before first-line ARSI treatment, while three patients had received prior ARSI treatment before blood collection. EnhanceAR-Seq revealed that the most frequent genomic events detected were AR/enhancer alterations (copy number gain, tandem duplication or missense mutations) in 9 patients (45%), of which 5 patients had both AR gene body and enhancer copy number gain. The other 4 patients each had a single genomic event detected by EnhanceAR-Seq: AR amplification, AR enhancer amplification, AR and AR enhancer tandem duplication, and AR W742C single nucleotide variation. Cell-free DNA-detected alterations in the full AR locus including the AR enhancer were highly significant for inferior OS (P = 0.0009; HR = 17.0) but not for PFS (P = 0.2; HR = 2.2) by Kaplan-Meier analysis across all 20 patients. Subset analysis of the 17 patients with plasma analyzed prior to first-line ARSI treatment revealed that AR/enhancer alterations again predicted significantly worse OS with a median survival of 16.1 months vs. not-reached (P = 0.0009; HR = 14.1). Conclusions: AR locus alterations detected by EnhanceAR-seq in plasma cell-free DNA collected prior to ARSI administration correlated with significantly worse overall survival in patients with mCRPC. If corroborated, our results suggest that AR/enhancer genomic alterations represent a potent pre-treatment prognostic biomarker in mCRPC patients. Citation Format: Pradeep Singh Chauhan, Steven H. Hartman, Ha X. Dang, Jace Webster, Haley Ellis, Wenjia Feng, Peter K. Harris, Elisa M. Ledet, Ellen B. Jaeger, Patrick J. Miller, Sydney A. Caputo, Russell K. Pachynski, Oliver Sartor, Christopher A. Maher, Aadel A. Chaudhuri. Cell-free DNA alterations in the AR/enhancer locus measured before AR signaling inhibition portend poor overall survival in metastatic castration resistant prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 550.

Background: Liquid biopsy analysis of cell-free DNA (cfDNA) and circulating tumor cells (CTCs) have emerged as potential tools to predict response to androgen receptor (AR)-directed therapy in metastatic prostate cancer (mPCa). However, current clinically available assays (e.g. CTC AR-V7) focus on one AR variant, demonstrate low sensitivity, and remain underutilized. The recent discovery of AR enhancer amplification in 81% of metastatic patients and its association with disease resistance opens opportunities to improve upon current assays. We hypothesized that tracking AR/enhancer genomic alterations in plasma cfDNA would more sensitively detect resistance with higher accuracy than the CTC AR-V7 assay used in clinical practice. Methods: We developed a targeted sequencing assay and analysis method called Enhancer and neighboring loci of Androgen Receptor Sequencing (EnhanceAR-Seq). We applied EnhanceAR-Seq to plasma collected from 40 patients with mPCa treated with AR-directed therapy to monitor AR/enhancer genomic alterations and correlated these events with therapy resistance, progression-free survival (PFS), and overall survival (OS). In parallel, the Oncotype DX AR-V7 Nucleus Detect CTC assay was performed in peripheral blood from a subset of patients. Results: The most frequent genomic events detected were AR/enhancer alterations (copy number variation, tandem duplication or missense mutations) in 18 patients (45%), of which 16 patients (40%) had AR enhancer copy number gain or tandem duplication. Three patients (8%) were found to have independent AR enhancer amplification without AR gene body amplification. TP53 (8 patients, 20%) and PTEN (6 patients, 15%) were the other most frequently altered genes, subject to nonsynonymous mutations and copy number alterations. We also detected the TMPRSS2-ERG gene fusion in 5 cases (13%). cfDNA-detected alterations in the full AR locus including the AR enhancer were highly significant for overall survival (P=0.0015; HR=11.5) by Kaplan-Meier analysis, as well as progression-free survival by Kaplan-Meier analysis (P=0.0002; HR=6.8) and multivariate Cox proportional hazards regression (P=0.001; HR=12.7). While AR/enhancer alterations in plasma cfDNA identified 18 out of 23 treatment-resistant cases with 100% positive predictive value and 78% sensitivity (Fisher's P0.99). Conclusions: cfDNA based AR locus alterations including the enhancer are strongly associated with resistance to AR-directed therapy and significantly worse survival outcomes. EnhanceAR-Seq significantly outperformed the CTC AR-V7 assay used in standard clinical practice. Citation Format: Pradeep S. Chauhan, Ha X. Dang, Haley Ellis, Wenjia Feng, Peter K. Harris, Grace Smith, Mark Qiao, Katherine Dienstbach, Rachel Beck, Andrew Atkocius, Faridi Qaium, Jingqin Luo, Jeff M. Michalski, Joel Picus, Russell K. Pachynski, Christopher A. Maher, Aadel A. Chaudhuri. Detection of cell-free DNA alterations in AR and its enhancer significantly outperformed CTC AR-V7 detection for identifying treatment resistance in patients with metastatic prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-269.

5529 Background: Predicting primary resistance to androgen receptor (AR)-directed therapies is critical for personalizing treatment of metastatic prostate cancer (mPCa). Analyses of liquid biopsies are potential tools but remained underutilized due to limited sensitivity. We developed a cell-free DNA (cfDNA) assay (EnhanceAR-Seq) to monitor genomic alterations in mPCa including AR enhancer duplication, a resistance marker recently discovered in ~81% of mPCa patients. Here we show that applying EnhanceAR-Seq to plasma cfDNA to monitor alterations of AR gene and enhancer ( AR/enhancer) predicted primary resistance with high sensitivity and outperformed the clinically validated CTC AR-V7 assay. Methods: Forty mPCa patients were prospectively enrolled at the Washington University School of Medicine Siteman Cancer Center with plasma cfDNA analyzed by EnhanceAR-Seq. Twenty-five of them also had the Oncotype DX AR-V7 Nucleus Detect CTC assay performed at a similar timepoint at the discretion of the treating oncologist. All patients received AR-directed therapy (eg. abiraterone, enzalutamide) and underwent standard-of-care clinical and laboratory follow-up. Primary resistance was defined as PSA progression, change of treatment or death within 4 months of treatment initiation, or radiographic progression within 6 months. Results: Median clinical follow up after diagnosis was 50 months. EnhanceAR-Seq detected alterations targeting AR/enhancer in 18 patients (45%), TP53 in 8 patients (20%), and PTEN in 6 patients (15%). We found that AR/enhancer alterations (copy gain, tandem duplication, and point mutation) in cfDNA were strongly predictive of primary resistance to AR-directed therapy (PPV = 100%, Sens. = 89%). Our assay outperformed the CTC AR-V7 assay, which was positive in only two patients (PPV = 50%, Sens. = 6%). Furthermore, patients with AR/enhancer alterations had significantly worse progression-free survival (P = 0.0015; HR = 11.5) and overall survival (P = 0.0002; HR = 6.8). Finally, serial cell-free DNA analysis of 10 patients showed that AR/enhancer copy number gain was maintained or acquired in 5 of 6 AR-resistant cases, and neutrality maintained in 4 of 4 AR-sensitive cases. Conclusions: cfDNA-based AR/enhancer locus genomic alterations could potentially be used to predict primary resistance to AR-directed therapy with higher sensitivity than the clinically validated CTC AR-V7 assay, be used for serial timepoint monitoring and guiding personalized clinical decision-making.

Metastasis-associated in colon cancer-1 (MACC1) is key in promoting tumor proliferation and invasion, and is mediated by the hepatocyte growth factor (HGF) and mesenchymal-epithelial transition factor. Previous reports have revealed that MACC1 is a novel oncogene that is expressed in various types of gastrointestinal cancer. The present study comprised of 174 patients who underwent curative surgery for colorectal cancer (CRC). The correlation between gene expression and clinical parameters of the patients was assessed. It was identified that patients exhibiting high MACC1 expression levels were statistically more susceptible to distant metastases and a poor prognosis, and those exhibiting low MACC1 expression showed improved disease-free and overall survival than those with high expression. Therefore, the present data indicates that MACC1 expression levels may present as a prognostic factor in CRC patients.

SUMMARY Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.

The risk of relapse associated with oestrogen receptor-positive early breast cancer persists for at least 15 years after diagnosis. Several large clinical trials have examined extended adjuvant endocrine therapy.The MA.17 trial demonstrated improved disease-free survival (DFS) with use of letrozole for 5 years after some years of tamoxifen and an overall survival advantage for this approach in women with node-positive oestrogen receptor-positive cancer at diagnosis. The subsequent adjuvant tamoxifen - to offer more? and adjuvant tamoxifen: longer against shorter trials demonstrated a DFS advantage for 10 years of tamoxifen over 5 years. The recently reported MA.17R trial randomized women who had already completed 5 years of aromatase inhibitor therapy with or without previous tamoxifen to further 5 years of letrozole or placebo. DFS was significantly improved in the extended letrozole group, quality of life was similar but bone fracture rates were higher. The absolute benefit in terms of reduced distant recurrences in these studies is modest, and tolerability and compliance challenges remain.Physicians and patients now have multiple evidence-based treatment options for women who complete 5 years of adjuvant endocrine therapy. Extended therapy with either tamoxifen or letrozole should be considered for all and decision based on menopausal status, individual risk, tolerance and magnitude of potential benefit.

Sleep disturbance is common in borderline personality disorder (BPD), yet the reasons for this association are not clear. The present study sought to extend prior studies by testing emotion dysregulation as a mechanism underlying this association. We administered self-report measures of sleep quality (SQ), emotion dysregulation, and borderline features to two cross-sectional samples of undergraduates (sample 1: N = 293; sample 2: N = 188). In addition to replicating prior studies linking BPD features to poor SQ, analyses indicated that BPD features related to emotion dysregulation were most strongly correlated with poorer global SQ across both samples. Further, bootstrapping techniques revealed indirect associations by total score on the Difficulties in Emotion Regulation Scale (DERS), as well as for several of its facets (lack of strategies, lack of emotional clarity, lack of awareness). We discuss the potential clinical relevance of emotion dysregulation in the treatment of co-occurring sleep problems in BPD.

Genetic sex typing of vertebrate animals is an essential technique for research on reproductive phenomena such as sex determination of embryonic tissues. Polymerase chain reaction amplification of genomic DNA segments in the Z and W sex chromosomes has been widely used as a standard laboratory method to determine genetic sex of the chicken (Gallus gallus domesticus). However, conventional protocols for PCR determination of avian sex typically involve tedious steps of genomic DNA isolation, which often require relatively large amounts of tissue samples, and the purity of genomic DNA specimens significantly affects PCR efficiency. Moreover, detection of sex chromosome-specific PCR products by gel electrophoresis is prone to misjudgment caused by amplification of contaminating genomic DNA segments derived from tissue or DNA samples as well as previously generated PCR products. Thus, the credibility of genetic sex typing by conventional PCR-based methods that measure the relative amounts of the end product DNA amplicons critically depends on several experimental steps that are potentially vulnerable to errors. Here, we describe an optimized protocol of chicken genetic sex typing by TaqMan real-time quantitative PCR amplification of markers on the sex chromosomes. This TaqMan sex typing method accurately quantifies relative amounts of the Z and W sex chromosome markers directly from only 0.5 to 2 μL of total blood lysate without nucleic acid purification. The real-time amplification curves of the quantitative PCR reaction readily distinguishe truly homozygous (ZZ) and heterozygous (ZW) sex chromosomes from contamination of the sex chromosomal DNA, ensuring highly credible sex determination. Thus, the TaqMan typing of chicken genetic sex has several advantageous features for high-throughput operation compared with conventional methods.

Summary PIK3CA, which encodes the p110α subunit of PI3K, is frequently mutated and oncogenic in breast cancer. PI3Kα inhibitors are in clinical development and despite promising early clinical activity, intrinsic resistance is frequent among patients. We have previously reported that residual downstream mTORC1 activity upon treatment with PI3Kα inhibitors drives resistance to these agents. However, the mechanism underlying this phenotype is not fully understood. Here we show that in cancer cells resistant to PI3Kα inhibition, PDK1 blockade restores sensitivity to these therapies. SGK1, which is activated by PDK1, contributes to the maintenance of residual mTORC1 activity through direct phosphorylation and inhibition of TSC2. Targeting either PDK1 or SGK1 prevents mTORC1 activation, restoring the antitumoral effects of PI3Kα inhibition in resistant cells., Graphical Abstract, Highlights • The PDK1-SGK1 axis can sustain mTORC1 activity upon PI3K/AKT suppression • SGK1 directly phosphorylates TSC2, resulting in mTORC1 activation • Targeting PDK1 sensitizes PIK3CA-mutant cells to PI3Kα inhibition, Castel et al. show that PDK1 activates SGK1 to phosphorylate and inhibit TSC2, contributing to maintenance of mTORC1 activity and resistance of PIK3CA-mutant cancer cells to PI3Kα inhibition. Targeting either PDK1 or SGK1 restores the sensitivity of these resistant cancer cells to PI3Kα inhibition.

Activating mutations of PIK3CA are the most frequent genomic alterations in estrogen receptor (ER)-positive breast tumors, and selective phosphatidylinositol 3-kinase α (PI3Kα) inhibitors are in clinical development. The activity of these agents, however, is not homogeneous, and only a fraction of patients bearing PIK3CA-mutant ER-positive tumors benefit from single-agent administration. Searching for mechanisms of resistance, we observed that suppression of PI3K signaling results in induction of ER-dependent transcriptional activity, as demonstrated by changes in expression of genes containing ER-binding sites and increased occupancy by the ER of promoter regions of up-regulated genes. Furthermore, expression of ESR1 mRNA and ER protein were also increased upon PI3K inhibition. These changes in gene expression were confirmed in vivo in xenografts and patient-derived models and in tumors from patients undergoing treatment with the PI3Kα inhibitor BYL719. The observed effects on transcription were enhanced by the addition of estradiol and suppressed by the anti-ER therapies fulvestrant and tamoxifen. Fulvestrant markedly sensitized ER-positive tumors to PI3Kα inhibition, resulting in major tumor regressions in vivo. We propose that increased ER transcriptional activity may be a reactive mechanism that limits the activity of PI3K inhibitors and that combined PI3K and ER inhibition is a rational approach to target these tumors.

Both abundant epidermal growth factor receptor (EGFR or ErbB1) and high activity of the phosphatidyl-inositol 3-kinase (PI3K)–Akt pathway are common and therapeutically targeted in triple-negative breast cancer (TNBC). However, activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs. We found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3. The phosphorylation of HER3 and EGFR in response to these treatments was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A). MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors. In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3 decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction). Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting.

Somatic mutations in PIK3CA, the gene encoding the α isoform of PI3K (p110α), are frequent in breast cancer. These mutations lead to constitutive activation of the PI3K/AKT/mTORC1 pathway and are a potential therapeutic target. Despite the encouraging activity shown by selective PI3Kα inhibitors in early clinical studies, many breast cancer patients with PIK3CA-mutated tumors remain insensitive to these agents. We have previously shown both in preclinical models and in patients undergoing treatment with PI3Kα inhibitors that intrinsic resistance to PI3Kα inhibition is associated with sustained mTORC1 activation despite full AKT blockade. To elucidate how tumor cells can activate mTORC1 bypassing the AKT node, we performed a synthetic lethal RNAi screening using a library against the human kinome and phosphatome. We found that knockdown of phosphoinositide-dependent kinase 1 (PDK1) sensitized resistant cell lines to PI3Kα blockade, preventing mTORC1 signaling. These results were also validated by either shRNA-mediated PDK1 genetic knockdown or using the selective PDK1 kinase inhibitor, GSK2334470. The combination with PI3Kα inhibition abolished mTORC1 activity and resulted in superior antitumor activity in vitro and in vivo. Mechanistically, the combination of GSK2334470 with PI3Kα inhibition decreased the phosphorylation of the transcription factor FOXO3 at its T32 residue, causing its nuclear translocation, occupancy at the promoters of its target genes, and increased transcription. Given that full suppression of AKT activity was already achieved with PI3Kα inhibition alone, we hypothesized that another kinase downstream of PDK1 is responsible for FOXO3 phosphorylation and is inhibited upon PDK1 blockade. We demonstrated that SGK1, an AGC kinase upstream of FOXO3 and downstream of PDK1, is responsible for mediating resistance to PI3Kα inhibitors by activating mTORC1 through the direct phosphorylation of the tumor suppressor TSC2. Consistently, we found that SGK1 mRNA and protein levels are higher in PIK3CA-mutant cells resistant to PI3Kα. Our results were further corroborated through the characterization of the biochemical properties of a first-in-class SGK1 kinase inhibitor. This compound, in combination with a PI3Kα inhibitor, phenocopied the effects of PDK1 inhibition in all the models tested. Our findings uncover a parallel signaling cascade triggered by PI3Kα pharmacological inhibition that reconciles the sustained mTORC1 activation with lack of AKT enzymatic activity. This offers a novel therapeutic approach for PIK3CA-mutant breast tumors intrinsically resistant to PI3Kα inhibition. Citation Format: Pau Castel, Haley Ellis, Ruzica Bago, Eneda Toska, Kannan Srinivasaraghavan, F Javier Carmona, Pedram Razavi, Chandra Verma, Maura Dickler, Sarat Chandarlapaty, Edi Brogi, Dario Alessi, José Baselga, Maurizio Scaltriti. PDK1 blockade overcomes intrinsic resistance to PI3Kα inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2107.

e14535Background: The immune checkpoint inhibitors have the reported class effects of inducing hypo- or hyperthyroidism in a small but significant number of patients. However, the reported rates of...

Sex of birds is genetically determined through inheritance of the ZW sex chromosomes (ZZ males and ZW females). Although the mechanisms of avian sex determination remains unknown, the genetic sex is experimentally reversible by in ovo exposure to exogenous estrogens (ZZ-male feminization) or aromatase inhibitors (ZW-female masculinization). Expression of various testis- and ovary-specific marker genes during the normal and reversed gonadal sex differentiation in chicken embryos has been extensively studied, but the roles of sex-specific epigenetic marks in sex differentiation are unknown. In this study, we show that a 170-nt region in the promoter of CYP19A1/aromatase, a key gene required for ovarian estrogen biosynthesis and feminization of chicken embryonic gonads, contains highly quantitative, nucleotide base-level epigenetic marks that reflect phenotypic gonadal sex differentiation. We developed a protocol to feminize ZZ-male chicken embryonic gonads in a highly quantitative manner by direct injection of emulsified ethynylestradiol into yolk at various developmental stages. Taking advantage of this experimental sex reversal model, we show that the epigenetic sex marks in the CYP19A1/aromatase promoter involving DNA methylation and histone lysine methylation are feminized significantly but only partially in sex-converted gonads even when morphological and transcriptional marks of sex differentiation show complete feminization, being indistinguishable from gonads of normal ZW females. Our study suggests that the epigenetic sex of chicken embryonic gonads is more stable than the morphologically or transcriptionally characterized sex differentiation, suggesting the importance of the nucleotide base-level epigenetic sex in gonadal sex differentiation.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal adult malignancy due to the propensity of this disease to metastasize. Circulating tumor cells (CTCs) are thought to be enriched for cells with metastatic potential and their characterization offers a means to understand the biological underpinnings of the distal spread of cancer. However, CTCs are rare cells in the blood and their isolation has posed a signifcant technological challenge. Multiple platforms have emerged in capturing these cells with most relying on a positive antibody based selection process (e.g. EpCAM). Our initial molecular characterization of pancreatic CTCs in the genetically engineered LSL-KrasG12D, Trp53flox/flox or +, Pdx1-Cre (KPC) mouse model utilized a microfluidic anti-EpCAM device followed by RNA sequencing to identify aberrant non-canonical WNT signaling in CTC populations (Yu M*, Ting DT*, et al. Nature 2012). Wnt2 was found to increase metastatic potential by enhancing anoikis resistance, a requirement of CTC survival, through activation of Tak1 kinase. However, this study was limited in only providing a partial CTC signature given the variable purity of these purified cell populations and analysis of bulk CTC populations could not provide the resolution to truly assess heterogeneity of these cells. Furthermore, a recent study has shown that cells from the mouse model may disseminate into circulation at an early point in PDAC development through epithelial-to-mesenchymal transition (EMT), which would generate CTCs with very low EpCAM expression and therefore would be missed in an EpCAM based capture device (Rhim AD et al. Cell 2012). We have overcome these barriers by employing a novel microfluidic isolation device to capture high numbers of CTCs that can be isolated as single cells (Ozkumur E*, Shah AM*, et al. Science Translational Medicine 2013). This device achieves high efficiency negative depletion of normal blood cells providing an enriched population of CTCs in solution that are not biased by a particular extracellular epitope and without any antibody interactions that could affect expression profiles. This has provided a method to truly understand the key transcriptional programs that differentiate CTCs from their primary tumors on an isogenic mouse background. Five tumor-bearing KPC mice generated a total of 168 single CTCs that were morphologically intact and subjected to a modified single cell RNA sequencing protocol (Tang F et al., Nature Protocols 2010). A total of 75 (45%) of these single CTCs were of sufficient quality for RNA sequencing indicating that the majority (55%) of intact CTCs selected likely had lost viability in the process of hematogenous transit. Single cell RNA-sequencing was also performed on 12 normal leukocytes (WBCs) from a control mouse, 12 mouse embryonic fibroblasts (MEFs), 16 single cells from the mouse NB508 pancreatic cancer cell line, and 34 (min 8 replicates) samples from primary tumors matched to the CTCs. Unsupervised hierarchical clustering of single cell samples demonstrated clear separation of MEFs, the NB508 pancreatic cancer cell line, and normal WBCs supporting the technical validity of the sequencing approach. Analysis of candidate CTCs identified three major CTC clusters, which were all distinct form matched primary tumors as well as from the NB508 cancer cell line. The must abundant CTC cluster comprised 41 of 75 cells (55%) and was defined by presence of epithelial markers (Krt7, Krt8, Krt18, Krt19) consistent with a “classical” CTC phenotype (CTC-c). The second CTC cluster was defined by enrichment of platelet markers CD41 (Itga2b) and CD61 (Itgb3) (CTC-plt) and a third having enrichment of cellular proliferation genes including Mki67 (CTC-pro). Single cell heterogeneity was assessed by intra-cluster correlation coeffficients, where lower values reflect higher heterogeneity. Not surprisingly, single cell heterogeneity was much higher in CTCs (mean correlation coefficient 0.42, 95% CI 0.36-0.47) compared to cancer cell lines (mean 0.86, 95% CI 0.80-0.91, p-value 1.2 x 10-15), but was notably similar comparing CTCs to single primary tumor cells (mean 0.38, 95% CI 0.28-0.47). Focusing on the dominant CTC-c cells, we used a non-parametric differential gene expression analysis including a rank product (RP) methodology suitable for large variations in absolute transcript levels found in single cell expression data (Breitling R et al. FEBS Letters 2004). Using a stringent FDR of ≤ 0.01, CTC-c cells had 878 genes with increased expression and 774 genes with reduced expression when compared with matched primary tumors. CTC-c cells were enriched for MAPK, as well as WNT, TGF-β, Neurotrophin, Toll-like receptor, and B-cell receptor signaling pathways. Analysis of a panel of EMT genes with significant differential expression revealed that CTC-c cells were in a biphenotypic state with universal loss of the epithelial markers E-cadherin (Cdh1) and Muc1, while the mesenchymal genes Cdh11 and Vim were found to be expressed much more heterogeneously amongst individual CTCs. Proposed pancreatic cancer stem cell genes were also evaluated and the Aldh1a1 and Aldh1a2 genes were found to be significantly enriched in CTC-c. Expression of both Aldh1a1 and Aldh1a2 in matched primary tumors was done through RNA in situ hybridization (RNA-ISH) revealing a heterogeneous distribution of these stem cell genes in both the stromal and epithelial compartments of the tumor. This highlighted the potential relevance of these stem cell markers in tumor cells dynamically shifting between epithelial and non-epithelial states. To provide further insight into the potential region from which CTCs emanate from the primary tumor, we selected the most highly enriched CTC transcripts found in ≥ 90% of all classical CTCs. Three genes met these criteria, which were decorin (Dcn), insulin-like growth factor binding protein 5 (Igfbp5), and Kruppel-like factor 4 (Klf4). Each of these genes has been previously implicated in pancreatic cancer development and were evaluated by RNA-ISH in primary tumor specimens to determine if they colocalized in particular tumor cells. Dcn is an extracellular matrix proteoglycan known to be expressed in a wide range of tumor stroma and by RNA-ISH was found primarily in the stromal elements of the tumor. However, both Igfbp5 and Klf4 were found to be focally expressed in cells at the epithelial-stromal interface. Although these genes are co-expressed in a minority of primary tumor cells, they are co-expressed at high levels in 85% of all classical CTCs. Together with the mixed epithelial/mesenchymal marks and enrichment of Aldh1a2 cells in stromal elements, these data point to the majority of viable CTCs emanating from the epithelial/stromal interface. In summary, we have successfully purified individual pancreatic CTCs using a novel microfluidic device and provided the first comprehensive single cell transcriptome analysis of these rare but exceptional cells. Three major classes of CTCs have been identified that would not have been possible without a single cell approach and we have characterized the major pathways that define these different subsets. The most abundant CTCs were found to have robust expression of keratin genes and are defined by a mixed E/M state with enrichment of Aldh1a1 and Aldh1a2 stem cell genes. These classical CTCs are marked by the co-expression of Igfbp5 and Klf4, which appear to localize to the epithelial-stromal interface in primary tumors. Ultimately, CTC cultures and functional testing will determine the contribution of these genes to CTC metastatic potential. This deep analysis of CTCs at single cell resolution has provided new biological insight into the metastatic cascade that will inform the development of novel therapies to treat this deadly disease. Citation Format: David T. Ting, Ben S. Wittner, Ajay M. Shah, David T. Miyamoto, Brian W. Brannigan, Kristina Xega, Jordan Ciciliano, Olivia C. MacKenzie, Julie Trautwein, Mohammad Shahid, Haley L. Ellis, Na Qu, Nabeel Bardeesy, Miguel N. Rivera, Ravi Kapur, Sridhar Ramaswamy, Toshi Shioda, Mehmet Toner, Shyamala Maheswaran, Daniel A. Haber. Diversity of circulating tumor cells in a mouse pancreatic cancer model identified by single cell RNA sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr NG04. doi:10.1158/1538-7445.AM2014-NG04

Activating mutations of PIK3CA, the gene encoding the p110α subunit of PI3K, are frequent in breast cancer and selective inhibitors of this enzyme have shown promising clinical activity in breast tumors harboring these mutations. We studied the case of a patient with metastatic breast cancer harboring a PIK3CA mutation that was treated in a clinical trial with BYL719, a highly selective PI3Kα inhibitor. The treatment resulted in a robust partial response that lasted 7 months followed by rapid progression and death. A rapid autopsy was performed with collection of tissue samples from 16 different metastatic sites. We compared by whole genome and exome sequencing the original primary tumor, a rapidly progressing lung metastasis and a periaortic lesion that was still responding to BYL719 at time of death. Besides several common alterations, PTEN loss and a missense mutation were detected only in the lung metastasis. Using targeted exome sequencing we analyzed all the other available samples. Strikingly, we observed a consistent loss in PTEN (via different mechanisms such as deletion, splice site mutation and frameshift mutations) in all the lesions refractory to BYL719 but not in the responding ones. In every case, the loss of PTEN was also documented by lack of protein expression by immunohistochemistry (IHC). Finally, we were able to build a dendrogram showing the phylogenetic evolution of the lesions and the evolutionary convergence of the PTEN alterations. To validate PTEN loss as a possible mechanism of acquired resistance to selective PI3Kα inhibition, we generated doxycycline-inducible PTEN shRNA stable clones starting from three different BYL719-sensitive cell lines. In all the studied models, induction of PTEN shRNA resulted in resistance to BYL719. Since PTEN deficient genetic models have been shown to rely on the β subunit of the PI3K holoenzyme, we tested whether the concomitant inhibition of both p110α and p110β was sufficient to revert the resistant phenotype. BKM120 (a pan-PI3K inhibitor) or the addition of AZD6482 (p110β inhibitor) to BYL719 re-sensitized the cells to BYL719. To expand our findings in vivo, we generated a patient-derived xenograft (PDX) model from a PTEN-null non-responding lesion (lung). Consistently, this PDX model was refractory to the antitumor activity of BYL719 but conserved sensitivity to BKM120 or the combination of AZD6482 and BYL719. In both cases, IHC analysis revealed a decrease in PI3K/AKT downstream effectors pPRAS40 (246) and pS6 (240/4) staining with BKM120 or AZD6482+BYL719, but not with BYL719 alone. Preliminary analyses of other samples collected from patients treated with BYL719 suggest that PTEN loss is a relatively frequent event upon therapy progression. Taken together, the different mechanisms that inactivate PTEN in the tumor treated with BYL719 can be explained by convergent phenotypic evolution in a heterogeneous tumor and highlight the importance of PTEN and PI3Kβ in acquired resistance to PI3Kα inhibitors. Citation Format: Pau Castel, Dejan Juric, Helen Won, Benjamin Ainscough, Haley Ellis, Saya Ebbesen, Malachi Griffith, Obi Griffith, Iyer Gopakumar, Dennis Sgroi, Steven Isakoff, Elaine Mardis, David Solit, Scott Lowe, Cornelia Quadt, Malte Peters, Michael Berger, Maurizio Scaltriti, José Baselga. Loss of PTEN leads to clinical resistance to the PI3Kα inhibitor BYL719 and provides evidence of convergent evolution under selective therapeutic pressure. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-327. doi:10.1158/1538-7445.AM2014-LB-327