Clinical and molecular determinants of PSA response to bipolar androgen therapy in prostate cancer.

BACKGROUND: Bipolar androgen therapy (BAT) is a novel therapy known to be effective in a subset of men with metastatic castrate resistant prostate cancer (mCRPC). A better understanding of responders and nonresponders to BAT would be useful to clinicians considering BAT therapy for patients. Herein we analyze clinical and genetic factors in responders/nonresponders to better refine our understanding regarding which patients benefit from this innovative therapy. METHODS: mCRPC patients were assessed for response or no response to BAT. Patients with PSA declines of greater than 50% from baseline after 2 or more doses of testosterone were considered to be responders. Whereas, nonresponders had no PSA decline after 2 doses of testosterone and subsequently manifest a PSA increase of >50%. Differences between these two groups of patients were analyzed using clinical and laboratory parameters. All patients underwent genomic testing using circulating tumor DNA (ctDNA) and germline testing pre-BAT. RESULTS: Twenty five patients were nonresponders and 16 were responders. Baseline characteristics between nonresponders and responders varied. Responders were more likely to have had a radical prostatectomy as definitive therapy and were more likely to have been treated with an androgen receptor (AR) antagonist (enzalutamide or apalutamide) immediately before BAT (compared to abiraterone). Duration of prior enzalutamide therapy was longer in responders. Nonresponders were more likely to have bone-only metastases and responders were more likely to have nodal metastases. Assays detected ctDNA AR amplifications more often in responding patients. Responders trended toward having the presence of more TP53 mutations at baseline. CONCLUSIONS: BAT responders are distinct from nonresponders in several ways however each of these distinctions are imperfect. Patterns of metastatic disease, prior therapies, duration of prior therapies, and genomics each contribute to an understanding of patients that will or will not respond. Additional studies are needed to refine the parameters that clinicians can utilize before choosing among the numerous treatment alternatives available for CRPC patients.

Extrahepatic deficiency of transferrin receptor 2 is associated with increased erythropoiesis independent of iron overload.

Transferrin receptor 2 (TFR2) is a transmembrane protein expressed mainly in hepatocytes and in developing erythroid cells and is an important focal point in systemic iron regulation. Loss of TFR2 function results in a rare form of the iron-overload disease hereditary hemochromatosis. Although TFR2 in the liver has been shown to be important for regulating iron homeostasis in the body, TFR2's function in erythroid progenitors remains controversial. In this report, we analyzed TFR2-deficient mice in the presence or absence of iron overload to distinguish between the effects caused by a high iron load and those caused by loss of TFR2 function. Analysis of bone marrow from TFR2-deficient mice revealed a reduction in the early burst-forming unit-erythroid and an expansion of late-stage erythroblasts that was independent of iron overload. Spleens of TFR2-deficient mice displayed an increase in colony-forming unit-erythroid progenitors and in all erythroblast populations regardless of iron overload. This expansion of the erythroid compartment coincided with increased erythroferrone (ERFE) expression and serum erythropoietin (EPO) levels. Rescue of hepatic TFR2 expression normalized hepcidin expression and the total cell count of the bone marrow and spleen, but it had no effect on erythroid progenitor frequency. On the basis of these results, we propose a model of TFR2's function in murine erythropoiesis, indicating that deficiency in this receptor is associated with increased erythroid development and expression of EPO and ERFE in extrahepatic tissues independent of TFR's role in the liver.

Corepressor Rcor1 is essential for murine erythropoiesis.

The corepressor Rcor1 has been linked biochemically to hematopoiesis, but its function in vivo remains unknown. We show that mice deleted for Rcor1 are profoundly anemic and die in late gestation. Definitive erythroid cells from mutant mice arrest at the transition from proerythroblast to basophilic erythroblast. Remarkably, Rcor1 null erythroid progenitors cultured in vitro form myeloid colonies instead of erythroid colonies. The mutant proerythroblasts also aberrantly express genes of the myeloid lineage as well as genes typical of hematopoietic stem cells (HSCs) and/or progenitor cells. The colony-stimulating factor 2 receptor ß subunit (Csf2rb), which codes for a receptor implicated in myeloid cytokine signaling, is a direct target for both Rcor1 and the transcription repressor Gfi1b in erythroid cells. In the absence of Rcor1, the Csf2rb gene is highly induced, and Rcor1(-/-) progenitors exhibit CSF2-dependent phospho-Stat5 hypersensitivity. Blocking this pathway can partially reduce myeloid colony formation by Rcor1-deficient erythroid progenitors. Thus, Rcor1 promotes erythropoiesis by repressing HSC and/or progenitor genes, as well as the genes and signaling pathways that lead to myeloid cell fate.

The Corepressor Rcor1 Is Essential for Normal Myeloerythroid Lineage Differentiation.

Based on its physical interactions with histone-modifying enzymes, the transcriptional corepressor Rcor1 has been implicated in the epigenetic regulation blood cell development. Previously, we have demonstrated that Rcor1 is essential for the maturation of definitive erythroid cells and fetal survival. To determine the functional role of Rcor1 in steady-state hematopoiesis in the adult, we used a conditional knockout approach. Here, we show that the loss of Rcor1 expression results in the rapid onset of severe anemia due to a complete, cell autonomous block in the maturation of committed erythroid progenitors. By contrast, both the frequency of megakaryocyte progenitors and their capacity to produce platelets were normal. Although the frequency of common lymphoid progenitors and T cells was not altered, B cells were significantly reduced and showed increased apoptosis. However, Rcor1-deficient bone marrow sustained normal levels of B-cells following transplantation, indicating a non-cell autonomous requirement for Rcor1 in B-cell survival. Evaluation of the myelomonocytic lineage revealed an absence of mature neutrophils and a significant increase in the absolute number of monocytic cells. Rcor1-deficient monocytes were less apoptotic and showed ∼100-fold more colony-forming activity than their normal counterparts, but did not give rise to leukemia. Moreover, Rcor1(-/-) monocytes exhibited extensive, cytokine-dependent self-renewal and overexpressed genes associated with hematopoietic stem/progenitor cell expansion including Gata2, Meis1, and Hoxa9. Taken together, these data demonstrate that Rcor1 is essential for the normal differentiation of myeloerythroid progenitors and for appropriately regulating self-renewal activity in the monocyte lineage.

HCMV infection of humanized mice after transplantation of G-CSF-mobilized peripheral blood stem cells from HCMV-seropositive donors.

Human cytomegalovirus (HCMV) infection, including primary infection resulting from transmission from a seropositive donor to a seronegative recipient (D(+)/R(-)), remains a significant problem in the setting of peripheral blood stem cell transplantation (PBSCT). The lack of a suitable animal model for studying HCMV transmission after PBSCT is a major barrier to understanding this process and, consequently, developing novel interventions to prevent HCMV infection. Our previous work demonstrated that human CD34(+) progenitor cell-engrafted NOD-scid IL2Rγc(null) (NSG) mice support latent HCMV infection after direct inoculation and reactivation after treatment with granulocyte colony-stimulating factor. To more accurately recapitulate HCMV infection in the D(+)/R(-) PBSCT setting, granulocyte colony-stimulating factor-mobilized peripheral blood stem cells from seropositive donors were used to engraft NSG mice. All recipient mice demonstrated evidence of HCMV infection in liver, spleen, and bone marrow. These findings validate the NSG mouse model for studying HCMV transmission during PBSCT.

A novel human cytomegalovirus locus modulates cell type-specific outcomes of infection.

Clinical strains of HCMV encode 20 putative ORFs within a region of the genome termed ULb' that are postulated to encode functions related to persistence or immune evasion. We have previously identified ULb'-encoded pUL138 as necessary, but not sufficient, for HCMV latency in CD34+ hematopoietic progenitor cells (HPCs) infected in vitro. pUL138 is encoded on polycistronic transcripts that also encode 3 additional proteins, pUL133, pUL135, and pUL136, collectively comprising the UL133-UL138 locus. This work represents the first characterization of these proteins and identifies a role for this locus in infection. Similar to pUL138, pUL133, pUL135, and pUL136 are integral membrane proteins that partially co-localized with pUL138 in the Golgi during productive infection in fibroblasts. As expected of ULb' sequences, the UL133-UL138 locus was dispensable for replication in cultured fibroblasts. In CD34+ HPCs, this locus suppressed viral replication in HPCs, an activity attributable to both pUL133 and pUL138. Strikingly, the UL133-UL138 locus was required for efficient replication in endothelial cells. The association of this locus with three context-dependent phenotypes suggests an exciting role for the UL133-UL138 locus in modulating the outcome of viral infection in different contexts of infection. Differential profiles of protein expression from the UL133-UL138 locus correlated with the cell-type dependent phenotypes associated with this locus. We extended our in vitro findings to analyze viral replication and dissemination in a NOD-scid IL2Rγ(c) (null)-humanized mouse model. The UL133-UL138(NULL) virus exhibited an increased capacity for replication and/or dissemination following stem cell mobilization relative to the wild-type virus, suggesting an important role in viral persistence and spread in the host. As pUL133, pUL135, pUL136, and pUL138 are conserved in virus strains infecting higher order primates, but not lower order mammals, the functions encoded likely represent host-specific viral adaptations.

Fancd2-/- mice have hematopoietic defects that can be partially corrected by resveratrol.

Progressive bone marrow failure is a major cause of morbidity and mortality in human Fanconi Anemia patients. In an effort to develop a Fanconi Anemia murine model to study bone marrow failure, we found that Fancd2(-/-) mice have readily measurable hematopoietic defects. Fancd2 deficiency was associated with a significant decline in the size of the c-Kit(+)Sca-1(+)Lineage(-) (KSL) pool and reduced stem cell repopulation and spleen colony-forming capacity. Fancd2(-/-) KSL cells showed an abnormal cell cycle status and loss of quiescence. In addition, the supportive function of the marrow microenvironment was compromised in Fancd2(-/-) mice. Treatment with Sirt1-mimetic and the antioxidant drug, resveratrol, maintained Fancd2(-/-) KSL cells in quiescence, improved the marrow microenvironment, partially corrected the abnormal cell cycle status, and significantly improved the spleen colony-forming capacity of Fancd2(-/-) bone marrow cells. We conclude that Fancd2(-/-) mice have readily quantifiable hematopoietic defects, and that this model is well suited for pharmacologic screening studies.

Apoptosis-stimulating protein of p53 (ASPP2) heterozygous mice are tumor-prone and have attenuated cellular damage-response thresholds.

The expression of ASPP2 (53BP2L), a proapoptotic member of a family of p53-binding proteins, is frequently suppressed in many human cancers. Accumulating evidence suggests that ASPP2 inhibits tumor growth; however, the mechanisms by which ASPP2 suppresses tumor formation remain to be clarified. To study this, we targeted the ASPP2 allele in a mouse by replacing exons 10-17 with a neoR gene. ASPP2(-/-) mice were not viable because of an early embryonic lethal event. Although ASPP2(+/-) mice appeared developmentally normal, they displayed an increased incidence of a variety of spontaneous tumors as they aged. Moreover, gamma-irradiated 6-week-old ASPP2(+/-) mice developed an increased incidence of high-grade T cell lymphomas of thymic origin compared with ASPP2(+/+) mice. Primary thymocytes derived from ASPP2(+/-) mice exhibited an attenuated apoptotic response to gamma-irradiation compared with ASPP2(+/+) thymocytes. Additionally, ASPP2(+/-) primary mouse embryonic fibroblasts demonstrated a defective G(0)/G(1) cell cycle checkpoint after gamma-irradiation. Our results demonstrate that ASPP2 is a haploinsufficient tumor suppressor and, importantly, open new avenues for investigation into the mechanisms by which disruption of ASPP2 pathways could play a role in tumorigenesis and response to therapy.

BMP4 regulates the hematopoietic stem cell niche.

Bone morphogenetic protein 4 (BMP4) is required for mesoderm commitment to the hematopoietic lineage during early embryogenesis. However, deletion of BMP4 is early embryonically lethal and its functional role in definitive hematopoiesis is unknown. Consequently, we used a BMP4 hypomorph to investigate the role of BMP4 in regulating hematopoietic stem cell (HSC) function and maintaining steady-state hematopoiesis in the adult. Reporter gene expression shows that Bmp4 is expressed in cells associated with the hematopoietic microenvironment including osteoblasts, endothelial cells, and megakaryocytes. Although resting hematopoiesis is normal in a BMP4-deficient background, the number of c-Kit+, Sca-1+, Lineage- cells is significantly reduced. Serial transplantation studies reveal that BMP4-deficient recipients have a microenvironmental defect that reduces the repopulating activity of wild-type HSCs. This defect is even more pronounced in a parabiosis model that demonstrates a profound reduction in wild-type hematopoietic cells within the bone marrow of BMP4-deficient recipients. Furthermore, wild-type HSCs that successfully engraft into the BMP4-deficient bone marrow show a marked decrease in functional stem cell activity when tested in a competitive repopulation assay. Taken together, these findings indicate BMP4 is a critical component of the hematopoietic microenvironment that regulates both HSC number and function.

Myelomonocytic cells are sufficient for therapeutic cell fusion in liver.

Liver repopulation with bone marrow-derived hepatocytes (BMHs) can cure the genetic liver disease fumarylacetoacetate hydrolase (Fah) deficiency. BMHs emerge from fusion between donor bone marrow-derived cells and host hepatocytes. To use such in vivo cell fusion efficiently for therapy requires knowing the nature of the hematopoietic cells that fuse with hepatocytes. Here we show that the transplantation into Fah(-/-) mice of hematopoietic stem cells (HSCs) from lymphocyte-deficient Rag1(-/-) mice, lineage-committed granulocyte-macrophage progenitors (GMPs) or bone marrow-derived macrophages (BMMs) results in the robust production of BMHs. These results provide direct evidence that committed myelomonocytic cells such as macrophages can produce functional epithelial cells by in vivo fusion. Because stable bone marrow engraftment or HSCs are not required for this process, macrophages or their highly proliferative progenitors provide potential for targeted and well-tolerated cell therapy aimed at organ regeneration.

FDA expedited approval and implications for rational formulary and health plan design.

DISCLOSURES: No funding supported this commentary. The authors are employed by Humana, Inc. Shrank reports board of directors work for GetWell Network and NCQA. The other authors have nothing to disclose.

MYC Promotes Bone Marrow Stem Cell Dysfunction in Fanconi Anemia.

Bone marrow failure (BMF) in Fanconi anemia (FA) patients results from dysfunctional hematopoietic stem and progenitor cells (HSPCs). To identify determinants of BMF, we performed single-cell transcriptome profiling of primary HSPCs from FA patients. In addition to overexpression of p53 and TGF-ß pathway genes, we identified high levels of MYC expression. We correspondingly observed coexistence of distinct HSPC subpopulations expressing high levels of TP53 or MYC in FA bone marrow (BM). Inhibiting MYC expression with the BET bromodomain inhibitor (+)-JQ1 reduced the clonogenic potential of FA patient HSPCs but rescued physiological and genotoxic stress in HSPCs from FA mice, showing that MYC promotes proliferation while increasing DNA damage. MYC-high HSPCs showed significant downregulation of cell adhesion genes, consistent with enhanced egress of FA HSPCs from bone marrow to peripheral blood. We speculate that MYC overexpression impairs HSPC function in FA patients and contributes to exhaustion in FA bone marrow.

Axially chiral P-N ligands for the copper catalyzed beta-borylation of alpha,beta-unsaturated esters.

The synthesis and resolution of a new axially chiral Quinazolinap ligand are reported. The application of this and other related P-N ligands to the copper catalyzed beta-borylation of alpha,beta-unsaturated esters resulted in conversions of up to 100% and ee values of up to 79%. A diastereomerically pure palladacycle of the new ligand was characterised by X-ray crystallography.

LMTK3 is essential for oncogenic KIT expression in KIT-mutant GIST and melanoma.

Certain cancers, including gastrointestinal stromal tumor (GIST) and subsets of melanoma, are caused by somatic KIT mutations that result in KIT receptor tyrosine kinase constitutive activity, which drives proliferation. The treatment of KIT-mutant GIST has been revolutionized with the advent of KIT-directed cancer therapies. KIT tyrosine kinase inhibitors (TKI) are superior to conventional chemotherapy in their ability to control advanced KIT-mutant disease. However, these therapies have a limited duration of activity due to drug-resistant secondary KIT mutations that arise (or that are selected for) during KIT TKI treatment. To overcome the problem of KIT TKI resistance, we sought to identify novel therapeutic targets in KIT-mutant GIST and melanoma cells using a human tyrosine kinome siRNA screen. From this screen, we identified lemur tyrosine kinase 3 (LMTK3) and herein describe its role as a novel KIT regulator in KIT-mutant GIST and melanoma cells. We find that LMTK3 regulated the translation rate of KIT, such that loss of LMTK3 reduced total KIT, and thus KIT downstream signaling in cancer cells. Silencing of LMTK3 decreased cell viability and increased cell death in KIT-dependent, but not KIT-independent GIST and melanoma cell lines. Notably, LMTK3 silencing reduced viability of all KIT-mutant cell lines tested, even those with drug-resistant KIT secondary mutations. Furthermore, targeting of LMTK3 with siRNA delayed KIT-dependent GIST growth in a xenograft model. Our data suggest the potential of LMTK3 as a target for treatment of patients with KIT-mutant cancer, particularly after failure of KIT TKIs.

Catheter ablation for failed surgical maze: comparison of cut and sew vs. non-cut and sew maze.

PURPOSE: To compare findings in patients undergoing atrial fibrillation(AF) and/or atrial flutter(AFl) ablation after failed cut and sew (CS) vs. non-cut and sew (NCS) surgical maze. METHODS: We compared 10 patients with prior CS to 25 with prior NCS maze undergoing catheter ablation after failed maze. RESULTS: Patient demographics: Age 68.3 ± 8.7 CS vs. 68.2 ± 9.2 NCS(P = 0.977), male 70% CS vs. 72% NCS(P = 1.000), LA size 5.11 ± 0.60 cm CS vs. 4.54 ± 0.92 cm NCS(P = 0.096), sternotomy 100% CS vs. 64% of NCS(P = 0.036). Concomitant heart surgery in 100% CS and 68% NCS(P = 0.073). NCS used radiofrequency 84%, cryoablation 8%, microwave 4%, and ultrasound 4%. All maze operations targeted pulmonary vein (PV) isolation. The maze also targeted the mitral isthmus 100% CS vs. 36% NCS(P = 0.001) and the tricuspid isthmus 90% CS vs. 40% NCS (P = 0.018). Maze failure arrhythmia mechanism was AF 0% CS and 56% NCS (P = 0.0006). Nine CS pts failed for AFl and 1 for RA tachycardia. For NCS pts, 11 failed for AFl. CS isolated 94% of PVs and NCS isolated only 26% of PVs (P < 0.0005). At EPS, clinical and induced arrhythmias were ablated and non-isolated PVs were isolated. After final ablation, arrhythmia-free rates were 60% for CS and 52% for NCS (P = 0.723) after 2.99 ± 2.35 years. CONCLUSIONS: After failed surgical maze, CS isolated nearly all PVs and NCS never isolated all PVs and the clinical rhythm was more frequently AF for NCS and AFl for CS. CS remains the surgical gold standard for durable PV isolation.

Pharmacy management strategies for improving drug adherence.

BACKGROUND: Significant gaps in quality pervade U.S. health care, leading to suboptimal care and rising costs. One key factor driving the apparent quality gaps and rising costs in the current health care system is the issue of nonadherence to prescription medications. OBJECTIVE: To describe quality gaps in managed care that are driven by nonadherence to prescription medications and characterize the components of successful pharmacy management strategies for overcoming nonadherence. SUMMARY: Collaborative networks and medication therapy management (MTM) programs are 2 pharmacy management initiatives that are useful in reducing medication nonadherence among plan members. The Pharmacy Quality Alliance has laid the foundation for developing useful pharmacy quality metrics, aggregating data, and reporting to both consumers and pharmacies. At the same time, the National Committee for Quality Assurance has developed MTM measures to monitor pharmacy quality. Both organizations have used Medicare Part D as an impetus for these initiatives in an effort to assess the value of the high-cost investment in prescription drugs resulting from the government mandate. CONCLUSION: Managed care stakeholders should strive toward a valuebased health care system by investing more on appropriate medication use, including initiatives to reduce nonadherence and avoid the high costs of treating severe disease in the future.

Atrial fibrillation ablation using very short duration 50 W ablations and contact force sensing catheters.

PURPOSE: The optimal radiofrequency (RF) power and lesion duration using contact force (CF) sensing catheters for atrial fibrillation (AF) ablation are unknown. We evaluate 50 W RF power for very short durations using CF sensing catheters during AF ablation. METHODS: We evaluated 51 patients with paroxysmal (n = 20) or persistent (n = 31) AF undergoing initial RF ablation. RESULTS: A total of 3961 50 W RF lesions were given (average 77.6 ± 19.1/patient) for an average duration of only 11.2 ± 3.7 s. As CF increased from < 10 to > 40 g, the RF application duration decreased from 13.7 ± 4.4 to 8.6 ± 2.5 s (p < 0.0005). Impedance drops occurred in all ablations, and for patients in sinus rhythm, there was loss of pacing capture during RF delivery suggesting lesion creation. Only 3% of the ablation lesions were at < 5 g and 1% at > 40 g of force. As CF increased, the force time integral (FTI) increased from 47 ± 24 to 376 ± 102 gs (p < 0.0005) and the lesion index (LSI) increased from 4.10 ± 0.51 to 7.63 ± 0.50 (p < 0.0005). Both procedure time (101 ± 19.7 min) and total RF energy time (895 ± 258 s) were very short. For paroxysmal AF, the single procedure freedom from AF was 86% at 1 and 2 years. For persistent AF, it was 83% at 1 year and 72% at 2 years. There were no complications. CONCLUSIONS: Short duration 50 W ablations using CF sensing catheters are safe and result in excellent long-term freedom from AF for both paroxysmal and persistent AF with short procedure times and small amounts of total RF energy delivery.

Case report: PET/CT, a cautionary tale.

BACKGROUND: The use of combined positron emission tomography/computerised tomography (PET/CT) scanners in oncology has been shown to improve the staging of tumours and the detection of relapses. However, mis-registration errors are increasingly recognised to be a common pitfall of PET/CT studies. CASE PRESENTATION: We report a patient with a germ cell tumour of the testis, who underwent a PET/CT scan to detect the site of relapse with a view to surgical removal. However, the PET/CT scan mislocalised the tumour site to be within the T2 vertebral body. A subsequent endoscopic ultrasound scan however showed the tumour to be anterior to the vertebral body, which was confirmed at surgery. CONCLUSION: In this report, we highlight the artefactual mislocalisation errors which may occur with PET/CT imaging, and the need to review and verify these scans.

AHRQ's Effective Health Care Program: its impact on managed care.

OBJECTIVE: To describe some of the managed care perspectives regarding the data development and coverage issues. BACKGROUND: Section 1013 of the Medicare Modernization Act of 2003 has initiated the formation of the Agency for Healthcare Research and Quality (AHRQ) Effective Health Care Program, which is evaluating the treatments for rheumatoid arthritis (RA) in the Medicare population. The results of these studies have the potential to impact future drug utilization. It is not known whether this data could be applied to the commercial population. SUMMARY: Payers (e.g., managed care organizations, pharmacy benefit managers) make decisions about which drugs will be covered and to which formulary "tier" the drug will be assigned. These decisions are made by evaluating current evidence based on safety, effectiveness, outcomes, and cost. Patients believe in a "warranty" of care, meaning that there will always be a treatment option whether they are compliant with their treatment regimen or not. All treatments are measured by a "value," and each stakeholder may see this value differently. A return on medical investment is one way to assess this value. CONCLUSIONS: Different stakeholders view treatment value in different ways. The evidence that will be identified through AHRQ's Effective Health Care Program will partially define this value. If this model succeeds, it has the potential to significantly affect health care.

The role of pharmacology in drug discovery.

The International Union of Pharmacology (IUPHAR) enthusiastically welcomes the decision by the Nature Publishing Group to launch its new journal, Nature Reviews Drug Discovery. The title of the new journal poses interesting questions for pharmacologists. Why 'Drug Discovery'? Would we have preferred 'Pharmacology'? And do these distinctions even matter, as aren't all pharmacologists involved somehow in drug discovery?