Search

Introduction Cisplatin, a platinum-based antineoplastic agent, is the cornerstone for the treatment of many malignancies. Nephrotoxicity is the primary dose-limiting toxicity, and various hydration regimens and supplementation strategies are used to prevent cisplatin-induced kidney injury. However, evidence-based recommendations on specific hydration regimens are limited. A systematic review was performed to evaluate clinical studies that have examined hydration and supplementation strategies to prevent cisplatin-induced nephrotoxicity. Materials and methods PubMed and Excerpta Medica databases were searched from 1966 through October 2015 for clinical trials and other studies focused on hydration regimens to prevent nephrotoxicity in cancer patients treated with cisplatin. The University of Oxford Centre for Evidence-Based Medicine criteria were used to grade level of evidence. Results Among the 1,407 identified studies, 24 were included in this systematic review. All studies differed on type, volume, and duration of hydration. Among the 24 studies, 5 evaluated short-duration hydration, 4 evaluated low-volume hydration, 4 investigated magnesium supplementation, and 7 reviewed forced diuresis with hydration. Short-duration and lower-volume hydration regimens are effective in preventing cisplatin-induced nephrotoxicity. Magnesium supplementation may have a role as a nephroprotectant, and forced diuresis may be appropriate in some patients receiving cisplatin. Conclusion Hydration is essential for all patients to prevent cisplatin-induced nephrotoxicity. Specifically, short-duration, low-volume, outpatient hydration with magnesium supplementation and mannitol forced diuresis (in select patients) represent best practice principles for the safe use of cisplatin. The Oncologist 2017;22:609-619 IMPLICATIONS FOR PRACTICE: The findings contained within this systematic review show that (a) hydration is essential for all patients to prevent cisplatin-induced nephrotoxicity, (b) short-duration, low-volume, outpatient hydration regimens appear to be safe and feasible, even in patients receiving intermediate- to high-dose cisplatin, (c) magnesium supplementation (8-16 milliequivalents) may limit cisplatin-induced nephrotoxicity, and (d) mannitol may be considered for high-dose cisplatin and/or patients with preexisting hypertension. These findings have broad implications for clinical practice and represent best practice principles for the prevention of cisplatin-induced nephrotoxicity.

Posterior reversible encephalopathy syndrome (PRES) is a clinical/radiological syndrome characterized by symptoms that can include seizure, headache, impaired vision and hypertension, and can be confirmed by magnetic resonance imaging. Numerous reports have emerged that describe PRES in cancer patients. The list of medications linked to PRES can include traditional cytotoxic chemotherapeutics (e.g., cisplatin, cyclophosphamide, and high-dose corticosteroids), newer agents that target the vascular endothelial growth factor pathway (e.g., bevacizumab, sunitinib, and pazopanib), and supportive care mediations (e.g., granulocyte colony stimulating factors and erythropoietin). We report, for the first time, a case of PRES that is secondary to treatment with enzalutamide, a potent androgen receptor antagonist used in the treatment of metastatic castration-resistant prostate cancer. Enzalutamide is approved for the treatment of both docetaxel-pretreated and chemotherapy-naïve metastatic castration-resistant prostate cancer. Enzalutamide has been previously linked to the increased risk of seizures. Clinicians should be aware that, in rare cases, patients treated with enzalutamide could potentially be at risk for PRES. If symptoms suggestive of PRES arise in patients receiving enzalutamide, the drug should be discontinued immediately and the diagnostic process should be initiated.

Purpose: Identify and summarize articles that describe the value that pharmacy residency training offers to sponsoring health systems. Summary: There is a tremendous gap between the number of resident applicants and the number of pharmacy residencies available. Informing health-system administration executives about the proven value of residency training is key to expanding the number of available positions. To address this disparity, a comprehensive and systematic literature search to identify publications highlighting the value that pharmacy residency training provides to the sponsor hospital or health system was conducted. Articles were identified through query of PubMed and SciVerse SCOPUS and through review of bibliographies from relevant articles. Twenty articles were identified and summarized in this annotated bibliography that demonstrate perceived and quantitative value of pharmacy residency training for health systems that sponsor residency training. Conclusion: Pharmacy residency training programs are essential for pharmacists that will primarily engage in direct patient care activities. This annotated bibliography includes key publications that provide evidence of the value that pharmacy residents provide to the sponsoring health system. This manuscript will aid prospective residency directors interested in developing new residency positions at new institutions or for residency program directors interested in expanding the total number of resident positions available at the existing sites.

Background Hypertension is one of the major side effect of VEGF-pathway inhibitors. There are no validated biomarkers to predict which cancer patients will develop hypertension. This study aimed to identify genetic predictors of hypertension induced by VEGF-pathway inhibitors. Methods A two-step, discovery-validation approach was used. The discovery set included 140 renal cell carcinoma patients from the TARGET study treated with sorafenib (400 mg twice daily) (PMID 30385613) and genotyped for 1,040 germline variants in 56 genes. The most statistically significant variant from the discovery set (chi-squared test) was tested in a validation set (cause-specific Cox model) consisting of 1,041 cancer patients treated with bevacizumab (10-15 mg/kg every two-three weeks) and genotyped using GWAS microarray platforms. For both studies, grade ≥2 hypertension (CTCAE v. 3.0) was used as the endpoint. Genetic associations were adjusted for age and sex. Results In the discovery set, the most statistically significant variant associated with hypertension in sorafenib-treated patients was rs444904 (G>A, P = 0.006). The A allele of rs444904 (minor allele frequency, MAF 0.14) increased the risk of hypertension. In the validation set, the A allele of rs427554 (G>A, in complete linkage disequilibrium with rs444904) increased the risk of hypertension in bevacizumab-treated patients (P = 0.008, MAF 0.11). rs444904 and rs427554 are intronic variants in PIK3R5. PIK3R5 encodes the regulatory subunit of PI3Kγ, which, when activated by VEGF, leads to nitric oxide (NO) production and vasodilation. These variants have been associated with decreased expression of PIK3R5 in blood (PMID 25954001), consistent with their effect in increasing the risk of hypertension. Conclusions Common genetic variants that could reduce PIK3R5 activity increase the risk of sorafenib- and bevacizumab-induced hypertension. In patients treated with these drugs, reduced activity or expression of PIK3R5 may lead to a reduction in NO production, resulting in vasoconstriction and hypertension. This study, for the first time, provides evidence for new predictive genetic markers of drug-induced hypertension that should be further evaluated for other VEGF-pathway inhibitors. Clinical trial identification TARGET CALGB 80303 NCT00088894 CALGB 40503 NCT00601900 CALGB 40502 NCT00785291 CALGB 90401 NCT00110214. Legal entity responsible for the study Federico Innocenti. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

305 Background: The Strata trial (NCT03061305) is a multi-institutional precision oncology collaboration structured as an observational protocol that aims to match patients to genomically-guided therapies. Methods: Selected University of North Carolina (UNC) metastatic prostate cancer (mPC) patients were enrolled on this IRB-approved study. Formalin fixed paraffin-embedded primary tumor specimens, without matched germline controls, were sent for targeted next generation sequencing (NGS) to detect actionable variants, including: mutations in 87 genes, copy number variations in 31 genes, and gene fusions in 46 gene drivers. mPC-related genes of particular interest included: AR, ATM, BRCA1/2, ERG, MSH2, MSH6, PTEN, RB1, and TP53. Results: Of the 92 cases sequenced, 5 [5%] failed testing. Of the 87 mPC patients (median age 69 years [47-86]) enrolled: 53 [61%] were white, 28 [32%] were black, 1 [1%] was Asian, and 5 [6%] declined to be identified. NGS data revealed 106 variants in 27 genes: 62 patients (71%) had at least one variant, 21 (24%) had 2 variants, 7 (8%) had 3 variants, and 4 (3%) had 4 variants. Among the 62 patients with at least 1 identified variant, TMPRSS2-ERG fusion occurred most frequently (50%), followed by TP53 (40%), and PTEN (16%). 6% of all sequenced patients had variants in DNA damage repair genes including ATM (3%), BRCA2 (2%) and MSH2 (1%). One patient had a SLC45A3-ERG fusion combined with PTEN deep deletion, which has been associated with a more aggressive phenotype. One patient with a microsatellite-instability high tumor was treated with pembrolizumab. Conclusions: The UNC experience shows that a high proportion of primary prostate cancer tumors from mPC patients have genomic variants, and one patient was treated based on these data. Limited actionability may reflect the landscape of currently FDA approved mPC treatments, and available clinical trials. It may also be due to a short follow-up, and these data could inform treatment planning upon progression.

458 Background: Fibroblast growth factor receptor (FGFR) inhibitors are a promising new targeted therapy for patients with metastatic urothelial cancer (UC) and FGFR alterations. FGFR-altered tumors are more likely to be of the luminal molecular subtype, which is less immune infiltrated and may be less likely to respond to immune checkpoint inhibitors (ICP). Methods: Metastatic UC patients at the University of North Carolina who underwent targeted exon sequencing (any CLIA-certified platform) and were treated with ICP since 2014 were identified. Patients with any FGFR alteration were compared to patients without alterations (including mutations, fusions, and amplifications in FGFR1-4). Overall response rates (ORR) to ICP were assessed by a radiologist (K.M.) per RECIST 1.1 and compared between FGFR-altered and unaltered tumors using Fisher’s exact tests. Patients who died prior to radiologic assessment were considered non-responders. Results: 66 patients (median age 70, 65% male, 76% white, 21% black) were identified. Most patients (74%) had received prior platinum-based chemotherapy, and 13% had received 2 or more prior lines of therapy. At the time of initiation of ICP, 32% of patients had a hemoglobin < 10, 33% had liver metastases, and 72% had a performance status > 0. Fifteen (22%) patients had FGFR alterations. The ORR for all patients was 15%, with ORR of 13% in FGFR-altered patients compared with 16% in unaltered patients (p = 1.0). No patients (0/9, 0%) with known pathogenic mutations in FGFR3 responded to ICP compared to 10/57 (18%) of patients without these alterations (p = 0.33). 46% of FGFR-altered patients who stopped ICP due to progression received subsequent therapy. Conclusions: Response rates to ICP are low and there was no difference in ORR between FGFR-altered and unaltered patients. While no patient with pathogenic FGFR3 mutations responded to ICP in our cohort, this difference did not reach statistical significance. Given low response rates overall, some FGFR-altered patients may benefit from treatment with FGFR inhibitors prior to ICP. Analysis of larger cohorts of patients as well as patients from clinical trials and more in-depth molecular profiling may add further clarity.

Background This phase 1/2 study evaluated the dose-limiting toxicity and maximum tolerated dose of MLN2704, a humanized monoclonal antibody MLN591 targeting prostate-specific membrane antigen, linked to the maytansinoid DM1 in patients with progressive metastatic castration-resistant prostate cancer. Patients and methods A total of 62 patients received MLN2704 at ascending doses on 4 schedules: weekly (60, 84, 118, and 165 mg/m2; 12 patients); every 2 weeks (120, 168, 236, and 330 mg/m2; 15 patients); every 3 weeks (330 and 426 mg/m2; 18 patients); and on days 1 and 15 of a 6-week schedule (6-week cycle, 330 mg/m2; 17 patients). The primary efficacy endpoint was a sustained ≥50% decline from baseline prostate-specific antigen (PSA) without evidence of disease progression. Toxicity, pharmacokinetics, immunogenicity, and antitumor activity were assessed. Results Neurotoxicity was dose-limiting. Overall, 44 patients (71%) exhibited peripheral neuropathy: 6 (10%) had grade 3/4. Neurotoxicity rates remained high despite increasing the dosing interval to 3 (13 of 14; one grade 3) and 6 weeks (16 of 17; three grade 3). MLN2704 pharmacokinetics were dose-linear. Rapid deconjugation of DM1 from the conjugated antibody was seen. In all, 5 patients (8%) experienced ≥50% decline in PSA; 5 (8%) had PSA stabilization lasting≥90 days. Only 2 of 35 patients on the 3- and 6-week schedules achieved a PSA decline of >50%. Conclusions MLN2704 has limited activity in metastatic castration-resistant prostate cancer. Disulfide linker lability and rapid deconjugation lead to neurotoxicity and a narrow therapeutic window.

Altered concentrations of ghrelin, motilin, and cholecystokinin (CCK) may contribute to gastric hypomotility. The aims of this study were to evaluate the concentrations of these hormones in patients tolerant and intolerant to gastric nutrition, assess the influence of prokinetic therapy on these hormone concentrations, determine the associations between these mediators and gastric emptying, and evaluate whether inflammation influences their concentrations.Post hoc analyses of 2 prospective studies that enrolled 20 critically ill patients with an aspirated gastric residual (GR)150 mL while receiving gastric enteral nutrition (intolerant group) and 10 critically ill patients with minimal GR (tolerant group). Patients with intolerance were also assessed 1 day after prokinetic therapy. Fasting serum concentrations of total ghrelin, acyl ghrelin (active), des-acyl ghrelin (inactive), motilin, CCK, and tumor necrosis factor (TNF)-α were determined. Gastric emptying was assessed concurrently using the acetaminophen absorption method.Compared to the tolerant group, the intolerant group had higher total ghrelin (1324.8 ± 1204.6 vs 285.1 ± 132.5 pg/mL; P.001), lower acyl ghrelin (70.5 ± 65.4 vs 208.5 ± 186.9 pg/mL; P.05), and lower acyl ghrelin to des-acyl ghrelin ratio (1.11 ± 1.35 vs 3.47 ± 3.21 pg/mL; P.05). Concentrations of other hormones and TNF-α were similar. Despite accelerated gastric emptying after prokinetic therapy, concentrations of all hormones and TNF-α were similar to baseline values. Hormone concentrations were not associated with gastric emptying or TNF-α.Patients intolerant to gastric nutrition generate less acyl ghrelin, which may contribute to gastric hypomotility. Intolerance is not associated with altered concentrations of other hormones. Hormone concentrations are not influenced by prokinetic therapy.

Purpose: To discover genetic determinants of overall survival (OS) in metastatic renal cell carcinoma (mRCC) patients treated with sorafenib. Patients and Methods: Information for 11,117 germline DNA variants from 56 genes was obtained, using both Illumina GoldenGate genotyping and imputation, from 295 patients originally enrolled in the phase III TARGET trial (155 sorafenib and 140 placebo patients). OS was this genotyping study's primary endpoint, and associations between variants and OS were tested using multivariate Cox regression. Associations were considered significant if P Results: Five intragenic variants significantly associated with OS. Among patients in the sorafenib arm, VEGFA rs1885657 (HR=17.3, 95% CI 5.7-52.7; P=1.4x10-4), ITGAV rs3816375 (HR=5.9, 95% CI 2.1-16.4; P=4.9x10-4), and WWOX rs8047917 (HR=4.1, 95% CI 1.9-8.8; P=3.3x10-4) associated with shorter OS. Among patients from both arms, FLT4 rs307826 (HR=13.8, 95% CI 3.0-62.6; P=1.2x10-4) and VEGFA rs3024987 (HR=3.0, 95% CI 1.7-5.4; P=8.3x10-5) associated with shorter OS. No significant associations were discovered in the placebo arm. Bioinformatic evidence was used to prioritize rs1885657, rs58159269 (in complete linkage disequilibrium with rs1885657), and rs307826. rs1885657 and rs58159269 increased luciferase activity in human endothelial (TIME, LPEC) and mRCC (Caki-1) cells, and rs58159269 increased cell proliferation/reduced sorafenib cytotoxicity, and reduced endothelial tube formation in TIME cells. rs307826 increased VEGFR-3 phosphorylation, and led to post-translational VEGFR-3 processing differences in HUVECs. Conclusions: Regulatory VEGFA and FLT4 variants associated with OS in mRCC patients induce cellular alterations that affect angiogenesis and sorafenib activity. These are novel and potentially impactful clinical biomarkers for mRCC and sorafenib, and their relevance to other multikinase inhibitors used in mRCC should be tested. Citation Format: Daniel James Crona, Andrew D. Skol, Veli-Matti Leppänen, Dylan M. Glubb, Amy S. Etheridge, Eleanor Hilliard, Carol Peña, Yuri K. Peterson, Nancy Klauber-DeMore, Kari K. Alitalo, Federico Innocenti. Clinical biomarkers of survival in renal cell carcinoma patients treated with sorafenib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1625.

Familial pulmonary arterial hypertension (PAH) is associated with mutations in bone morphogenetic protein type II receptor (BMPR2). Many of these mutations occur in the BMPR2 tail domain, leaving the SMAD functions intact. To determine the in vivo consequences of BMPR2 tail domain mutation, we created a smooth muscle-specific doxycycline-inducible BMPR2 mutation with an arginine to termination mutation at amino acid 899. When these SM22-rtTA x TetO7-BMPR2R899X mice had transgene induced for 9 wk, starting at 4 wk of age, they universally developed pulmonary vascular pruning as assessed by fluorescent microangiography. Approximately one-third of the time, the induced animals developed elevated right ventricular systolic pressures (RVSP), associated with extensive pruning, muscularization of small pulmonary vessels, and development of large structural pulmonary vascular changes. These lesions included large numbers of macrophages and T cells in their adventitial compartment as well as CD133-positive cells in the lumen. Small vessels filled with CD45-positive and sometimes CD3-positive cells were a common feature in all SM22-rtTA x TetO7-BMPR2R899X mice. Gene array experiments show changes in stress response, muscle organization and function, proliferation, and apoptosis and developmental pathways before RVSP increases. Our results show that the primary phenotypic result of BMPR2 tail domain mutation in smooth muscle is pulmonary vascular pruning leading to elevated RVSP, associated with early dysregulation in multiple pathways with clear relevance to PAH. This model should be useful to the research community in examining early molecular and physical events in the development of PAH and as a platform to validate potential treatments.

Reactivated androgen receptor (AR) signaling drives castration-resistant prostate cancer (CRPC). The novel AR targeting drugs abiraterone and enzalutamide have improved survival of CRPC patients. However, resistance to these agents develops and patients ultimately succumb to CRPC. Potential mechanisms of resistance include the following: 1) Expression of AR splice variants, such as the AR-V7 isoform, which lacks the ligand-binding domain; 2) AR missense mutations in the ligand-binding domain, such as F876L and T877A; and 3) Mutation or overexpression of androgen biosynthetic enzymes or glucocorticoid receptor. Several novel agents may overcome resistance mechanisms. Galeterone acts through multiple mechanisms that include degradation of AR protein and is being evaluated in CRPC patients positive for AR-V7. EPI-001 and related compounds inhibit AR splice variants by targeting the N-terminal transactivation domain of AR. Promising therapies and novel biomarkers, such as AR-V7, may lead to improved outcomes for CRPC patients.

Background: We propose that single-nucleotide polymorphisms (SNPs) in genes of the vascular endothelial growth factor pathway of angiogenesis will associate with survival in non-small-cell lung cancer (NSCLC) patients. Methods: Fifty-three SNPs in vascular endothelial growth factor-pathway genes were genotyped in 150 European stage I-III NSCLC patients and tested for associations with patient survival. Replication was performed in an independent cohort of 142 European stage I-III patients. Reporter gene assays were used to assess the effects of SNPs on transcriptional activity. Results: In the initial cohort, five SNPs associated (q < 0.05) with relapse-free survival (RFS). The minor alleles of intronic FLT1 SNPs, rs7996030 and rs9582036, associated with reduced RFS (hazard ratio [HR] = 1.67 [95% confidence interval, CI, 1.22-2.29] and HR = 1.51 [95% CI, 1.14-2.01], respectively) and reduced transcriptional activity. The minor alleles of intronic KRAS SNPs, rs12813551 and rs10505980, associated with increased RFS (HR = 0.64 [0.46-0.87] and HR = 0.64 [0.47-0.87], respectively), and the minor allelic variant of rs12813551 also reduced transcriptional activity. Lastly, the minor allele of the intronic KRAS SNP rs10842513 associated with reduced RFS (HR = 1.65 [95% CI, 1.16-2.37]). Analysis of the functional variants suggests they are located in transcriptional enhancer elements. The negative effect of rs9582036 on RFS was confirmed in the replication cohort (HR = 1.69 [0.99-2.89], p = 0.028), and the association was significant in pooled analysis of both cohorts (HR = 1.67 [1.21-2.30], p = 0.0001). Conclusions: The functional FLT1 variant rs9582036 is a prognostic determinant of recurrence in stage I-III NSCLC. Its predictive value should be tested in the adjuvant setting of stage I-III NSCLC.

Glucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and resistance to glucocorticoids in leukemia cells confers poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the prednisolone sensitivity of primary leukemia cells from 444 patients newly diagnosed with ALL and found significantly higher expression of CASP1 (encoding caspase 1) and its activator NLRP3 in glucocorticoid-resistant leukemia cells, resulting from significantly lower somatic methylation of the CASP1 and NLRP3 promoters. Overexpression of CASP1 resulted in cleavage of the glucocorticoid receptor, diminished the glucocorticoid-induced transcriptional response and increased glucocorticoid resistance. Knockdown or inhibition of CASP1 significantly increased glucocorticoid receptor levels and mitigated glucocorticoid resistance in CASP1-overexpressing ALL. Our findings establish a new mechanism by which the NLRP3-CASP1 inflammasome modulates cellular levels of the glucocorticoid receptor and diminishes cell sensitivity to glucocorticoids. The broad impact on the glucocorticoid transcriptional response suggests that this mechanism could also modify glucocorticoid effects in other diseases.

To review currently available literature on the oral multikinase inhibitor regorafenib and its role in the treatment of metastatic colorectal cancer (mCRC), and imatinib- and sunitinib-resistant gastrointestinal stromal tumors (GISTs).A comprehensive literature search was performed of PubMed/MEDLINE and American Society of Clinical Oncology (ASCO) abstracts (through August 2013).Preclinical pharmacological and phase I to III trials data analyzing regorafenib efficacy and safety in mCRC or imatinib- and sunitinib-resistant GIST patients were evaluated. All available English-language, peer-reviewed articles and ASCO abstracts with relevant information were reviewed.Regorafenib was approved for mCRC in September 2012 and for imatinib- and sunitinib-resistant GISTs in February 2013. Regorafenib is an inhibitor of stromal, angiogenic, and oncogenic receptor tyrosine kinases, as well as the RAF/MEK/ERK signaling pathway. Phase III CORRECT (Regorafenib Monotherapy for Previously Treated Metastatic Colorectal Cancer) trial data demonstrated an overall survival benefit for mCRC patients treated with regorafenib (6.4 vs 5.0 months; P = .0052). Phase III GRID (Gastrointestinal Stromal Tumors After Failure of Imatinib and Sunitinib) trial data revealed a progression-free survival benefit in imatinib- and sunitinib-resistant GIST patients (4.8 vs 0.9 months; P.0001). Its adverse event (AE) profile is comparable to that of other multikinase inhibitors. The most commonly observed grade ≥3 AEs included hypertension, hand-foot skin reaction, rash, diarrhea, and fatigue.Regorafenib is a novel oral multikinase inhibitor that has shown promising results for patients with advanced, unresectable or metastatic treatment-refractory CRCs or imatinib- and sunitinib-resistant GISTs.

For over 70 years, androgen deprivation therapy (ADT) has been a fundamental treatment paradigm for advanced prostate cancer.

The systemic treatment of cancer with traditional cytotoxic chemotherapeutic agents and more targeted agents is often complicated by the onset of adverse drug reactions. Pharmacogenetic prediction of adverse drug reactions might have consequences for dosing and efficacy. This review discusses relevant examples where the germline variant–toxicity relationship has been validated as an initial step in developing clinically useful pharmacogenetic markers and provides examples where germline variants have influenced dosing strategies and/or survival or other outcomes of efficacy. This review will also provide insight into the reasons why more pharmacogenetic markers have not been routinely integrated into clinical practice.

Background Pulmonary arterial hypertension (PAH) is thought to be driven by dysfunction of pulmonary vascular microendothelial cells (PMVEC). Most hereditary PAH is associated with BMPR2 mutations. However, the physiologic and molecular consequences of expression of BMPR2 mutations in PMVEC are unknown. Methods In vivo experiments were performed on adult mice with conditional endothelial-specific expression of the truncation mutation Bmpr2delx4+, with age-matched transactivator-only mice as controls. Phenotype was assessed by RVSP, counts of muscularized vessels and proliferating cells, and staining for thromboses, inflammatory cells, and apoptotic cells. The effects of BMPR2 knockdown in PMVEC by siRNA on rates of apoptosis were assessed. Affymetrix expression arrays were performed on PMVEC isolated and cultured from triple transgenic mice carrying the immortomouse gene, a transactivator, and either control, Bmpr2delx4+ or Bmpr2R899X mutation. Results Transgenic mice showed increased RVSP and corresponding muscularization of small vessels, with histologic alterations including thrombosis, increased inflammatory cells, increased proliferating cells, and a moderate increase in apoptotic cells. Expression arrays showed alterations in specific pathways consistent with the histologic changes. Bmpr2delx4+ and Bmpr2R899X mutations resulted in very similar alterations in proliferation, apoptosis, metabolism, and adhesion; Bmpr2delx4+ cells showed upregulation of platelet adhesion genes and cytokines not seen in Bmpr2R899X PMVEC. Bmpr2 mutation in PMVEC does not cause a loss of differentiation markers as was seen with Bmpr2 mutation in smooth muscle cells. Conclusions Bmpr2 mutation in PMVEC in vivo may drive PAH through multiple, potentially independent, downstream mechanisms, including proliferation, apoptosis, inflammation, and thrombosis.

Background While modulation of the serotonin transporter (5HTT) has shown to be a risk factor for pulmonary arterial hypertension for almost 40 years, there is a lack of in vivo data about the broad molecular effects of pulmonary inhibition of 5HTT. Previous studies have suggested effects on inflammation, proliferation, and vasoconstriction. The goal of this study was to determine which of these were supported by alterations in gene expression in serotonin transporter knockout mice. Methods Eight week old normoxic mice with a 5-HTT knock-out (5HTT-/-) and their heterozygote(5HTT+/-) or wild-type(5HTT+/+) littermates had right ventricular systolic pressure(RVSP) assessed, lungs collected for RNA, pooled, and used in duplicate in Affymetrix array analysis. Representative genes were confirmed by quantitative RT-PCR and western blot. Results RVSP was normal in all groups. Only 124 genes were reliably changed between 5HTT-/- and 5HTT+/+ mice. More than half of these were either involved in inflammatory response or muscle function and organization; in addition, some matrix, heme oxygenase, developmental, and energy metabolism genes showed altered expression. Quantitative RT-PCR for examples from each major group confirmed changes seen by array, with an intermediate level in 5HTT +/- mice. Conclusion These results for the first time show the in vivo effects of 5HTT knockout in lungs, and show that many of the downstream mechanisms suggested by cell culture and ex vivo experiments are also operational in vivo. This suggests that the effect of 5HTT on pulmonary vascular function arises from its impact on several systems, including vasoreactivity, proliferation, and immune function.

Idiopathic pulmonary arterial hypertension (IPAH) in human patients is associated with mutations in type 2 receptor for the bone morphogenic protein pathway (BMPR2). Mice expressing an inducible dominant negative form of BMPR2 in smooth muscle develop elevated right ventricular pressures when the transgene is activated. We hypothesized that transcriptional changes in these mice may allow insight into the early molecular events leading to IPAH. Microarray analysis was used to examine the transcriptional changes induced in whole lung by loss of normal smooth muscle cell (SMC) BMPR2 signaling in adult male or female mice (12 wk at time of death) expressing the transgene for either 1 or 8 wk. Our key results include a decrease in markers of smooth muscle differentiation, an increase in cytokines and markers of immune response, particularly in female mice, and a decrease in angiogenesis-related genes. These broad patterns of gene expression appear as early as 1 wk and are well established by 8 wk. Results were confirmed by quantitative RT-PCR to RNA from individual mice. Primary pulmonary artery SMC cultures transfected with small interfering RNA to BMPR2 also show loss of SMC markers myosin heavy chain 11 and calponin by quantitative RT-PCR and Western blot. These studies show classes of genes differentially regulated in response to loss of BMPR2 in SMC in vivo with clear relevance to the IPAH disease process, suggesting that the relevance of BMPR2 dysregulation may extend beyond proliferation.

Background: Sorafenib is an oral multikinase inhibitor that decreases tumor angiogenesis and proliferation. The antitumor efficacy and toxicity profiles of sorafenib vary among patients. Novel pathways and targets of sorafenib activity remain to be identified, and no predictive biomarkers of sorafenib activity exist to help guide clinicians. We aimed to identify novel genes associated with sorafenib activity by using an in vitro methodology based upon mouse genomics. Methods: We profiled primary mouse embryonic fibroblasts (MEFs) from 32 inbred strains for sorafenib cytotoxicity utilizing high content imaging and simultaneous evaluation of cell health parameters. The 32 strains were genomically characterized previously (PMID: 21623374). MEFs were treated with ten concentrations (0-300 μM) of sorafenib, incubated for 72 h, and then fixed and stained. Cytochrome C was immunolabeled in the fixed cells and imaged using an automated fluorescence microscope. An image analysis software assessed sorafenib-induced cytochrome C release from mitochondria. Dose response curves were generated from data, and EC50 values for each strain were identified using a Brain-Cousens model. Genome-wide association mapping, using the EMMA algorithm, was performed on cytochrome C EC50 values to identify quantitative trait loci (QTLs) associated with sorafenib cytotoxicity. Approximately 277,000 single nucleotide polymorphisms were tested, and genomic loci with p-values ≤ 1.0×10−7 were selected for additional analyses. Results: Interstrain EC50 variability among the 32 MEF strains was observed after 72 h (17.2-44.5 μM) sorafenib incubations. We identified one peak (chromosome 9 from 51-52 Mb; p = 1.0×10−8), which reached genome-wide significance and significantly associated with cytochrome C release. From this peak, we have identified candidate genes that may underlie variability in sorafenib-induced cytochrome C release from mitochondria. A total of nine genes expressed in MEF cells at mRNA level are present in this QTL. Of particular interest, the identified locus contains Rdx, a gene that encodes radixin. Radixin is a component of the ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) complex, and is conserved between humans and mice. In humans, radixin, as part of the EBP50 complex, has been shown to act as a tumor suppressor and to promote apoptosis in hepatocellular carcinoma by modulating β-catenin/E-cadherin (PMID: 23483729). It has also been shown to potentially enhance the metastatic potential of renal cell carcinoma (PMID: 20395446). Conclusions: Our high-throughput cellular genetics approach has detected robust interstrain cellular differences in sorafenib activity, and identified one region, which reached genome-wide significance, that potentially associates with sorafenib-induced cytochrome C release from mitochondria. Functional validation of Rdx and other promising candidates is currently ongoing. Citation Format: Daniel James Crona, Oscar Suzuki, O. Joseph Trask, Bethany Parks, Amber Frick, Timothy Wiltshire, Federico Innocenti. Identification of novel candidate genes associated with sorafenib cytotoxicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5486. doi:10.1158/1538-7445.AM2015-5486

Background: Sorafenib is an oral multikinase inhibitor, approved for hepatocellular, renal and thyroid carcinomas, which decreases tumor angiogenesis and proliferation. The antitumor efficacy and toxicity profiles of sorafenib vary among patients. No predictive biomarkers of sorafenib activity exist to help guide clinicians. Novel pathways and targets of sorafenib activity remain to be identified. We aimed to identify novel genes associated with sorafenib activity by using an in vitro methodology based upon mouse genomics. Methods: We profiled primary mouse embryonic fibroblasts (MEFs) from 32 inbred strains for sorafenib cytotoxicity utilizing high content imaging and simultaneous evaluation of cell health parameters. The 32 strains have been genomically characterized previously (PMID: 21623374). MEF cells were treated with varying concentrations (0-300 µM) of sorafenib, incubated for 24 h or 72 h, and then fixed and stained. Nuclear staining was used to assess sorafenib cytotoxicity and establish our cell viability phenotype. Dose response curves were generated from data, and EC50 values for each strain were identified using a Brain-Cousens model. Genome-wide association mapping, using the SNPster algorithm, was performed on cell viability EC50 values to identify quantitative trait loci (QTLs) associated with sorafenib cytotoxicity. Approximately 277,000 single nucleotide polymorphisms were tested, and genomic loci with p-values < 3.5x10-5 were selected for additional analyses. Results: Interstrain EC50 variability among the 32 MEF strains was observed after 24 h (21-121 µM) and 72 h (17-32 µM) sorafenib incubations. We identified three total peaks associated with cell viability: two on chromosome 13 (23 Mb apart; p = 3.4x10-5 and = 1.6x10-5, respectively), and one on chromosome 4 (p = 2.2x10-5). From these three peaks, we have identified candidate genes that may underlie variability in sorafenib cytotoxicity. A total of 16 genes expressed in MEF cells at mRNA level are present in these QTLs. Of particular interest, we identified one locus that contains Nfyc, a gene that encodes the C subunit of the NF-Y transcription factor. This transcription factor complex is conserved between humans and mice. In humans, NF-Y regulates MYC signaling and DNA-dependent transcription of PDGFR-β (a primary target of sorafenib) (PMID: 12167641). Conclusions: Our innovative high-throughput cellular genetics approach has identified three regions with genetic loci potentially associated with sorafenib cytotoxicity. This approach is capable of identifying robust interstrain cellular differences in sorafenib activity. Functional validation of Nyfc and other promising candidates should be conducted. Citation Format: Daniel J. Crona, Oscar Suzuki, O. Joseph Trask, Amber Frick, Bethany Parks, Tim Wiltshire, Federico Innocenti. A high-throughput cellular genetics approach to identifying genes associated with sorafenib response and toxicity. [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 5560. doi:10.1158/1538-7445.AM2014-5560