Citations

Abstract B-cell chronic lymphocytic leukemia (CLL) is an incurable disease and represents a significant health problem in the western world. We and others have reported that primary CLL B-cells spontaneously produce increased levels of proangiogenic basic fibroblast growth factor (bFGF) in vitro and that most CLL plasma contains elevated levels of bFGF. However, the precise role of bFGF in CLL pathobiology is not clearly understood. In this study we investigated the functional implication of the FGF/FGF receptor (FGFR) signaling axis in CLL B-cell biology. We have detected expression of FGFR1 and FGFR3 with comparatively higher levels of the latter receptor tyrosine kinase (RTK), but no or notably low levels of FGFR2/FGFR4, by flow cytometry and Western blot analyses in primary CLL B-cells. This observation was further supported by detection of FGFR1/FGFR3 transcripts in CLL B-cells by semi-quantitative reverse transcriptase polymerase chain reaction. Although both FGFR1 and FGFR3 in CLL B-cells remain as constitutively phosphorylated, we found significantly higher levels of phosphorylation on FGFR3 and thus this latter receptor is likely the predominant RTK of the FGFR family in these leukemic B-cells. Of note, in vitro stimulation of FGFRs with recombinant bFGF was unable to increase total phosphorylation on FGFRs from their constitutive basal levels in CLL B-cells. Further analysis using a bFGF neutralizing antibody suggested that FGFR phosphorylation in CLL B-cells is likely independent of bFGF ligation. We then interrogated the mechanism of how FGFRs were being phosphorylated and/or maintained at the observed constitutive levels of phosphorylation in CLL B-cells. Our previous studies established that Axl is a critical RTK in CLL B-cells since it acts as a docking site for multiple cellular kinases/lipase, an observation supported by earlier literatures in human malignancies. Given this, Axl is likely capable of cross talk with other RTKs including FGFRs to regulate FGFR-signaling in CLL B-cells. Therefore, in an effort to determine whether Axl is functionally associated with FGFR, we examined if these two RTKs exist in the same molecular complex in CLL B-cells. Indeed, immunoprecipitation assays demonstrated that Axl formed a complex with FGFR3 in CLL B-cells, suggesting that Axl is likely functionally linked to the FGFR signaling. In this regard we found that Axl inhibition, using a high-affinity Axl inhibitor (TP-0903; Tolero Pharmaceuticals), resulted in significant reduction of total FGFR phosphorylation in CLL B-cells. Additionally, siRNA-mediated partial depletion of Axl in CLL B-cells reduced total FGFR phosphorylation. In contrast, inhibition of FGFR phosphorylation using a high-affinity FGFR inhibitor could not alter phosphorylation levels on Axl RTK in CLL B-cells. Together, these findings suggest that Axl has a dominant role in the regulation of FGFR signaling in CLL B-cells. To find out if inhibition of FGFR can induce apoptosis in CLL B-cells we used a specific inhibitor for FGFR (TKI-258; Novartis) to treat CLL B-cells. Here we found a substantial level of apoptosis induction in the leukemic B-cells with a mean LD50 dose of ~2.5 μM. Interestingly, Axl inhibition by TP-0903 induced a robust level of apoptosis in CLL B-cells in the nanomolar dose range with a mean LD50 dose of 0.14 mM. Thus Axl inhibition exerts a very robust cytotoxic effect on CLL B-cell survival likely targeting both Axl and FGFR signaling pathways via Axl inhibition. In conclusion, we have detected expression of constitutively active FGFR1 and 3 in primary CLL B-cells and that inhibition of FGFR signaling induces considerable levels of CLL B-cell apoptosis albeit lower than that observed on Axl RTK inhibition. Interestingly, our findings here suggest that Axl forms an active RTK complex with FGFR and that Axl inhibition modifies FGFR phosphorylation levels. Thus it is likely that Axl RTK can regulate FGFR signaling in the CLL B-cells. In total these observations suggest that the finding of robust induction of apoptosis in CLL B-cells is as a result of targeting two signaling pathways with Axl inhibition: Axl and FGFR. These studies further support investigation of Axl inhibition as a way to develop a more effective and efficient therapeutic intervention for CLL patients. Disclosures Warner: Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Kay:Genetech: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

Abstract Introduction: The use of B-cell receptor (BCR) signal inhibitors-based therapies (e.g., Ibrutinib) for B-chronic lymphocytic leukemia (CLL) was initiated just a few years ago but has rapidly escalated due to their clinical efficacy and relative ease of use. However newer therapeutic approaches are needed due to multiple issues including the continued need to improve complete responses and reduce toxicity profiles. To that end our group has discovered a novel membrane target in the ubiquitous presence of Axl receptor tyrosine kinase (Axl RTK) on CLL B-cells and has reported that the Axl RTK inhibitor TP-0903 is able to induce apoptosis of CLL B-cells at nanomolar doses (Sinha, Clin Cancer Res, 2015). Given this we assessed if TP-0903 would be effective in the induction of apoptosis of leukemic B-cells from CLL patients who are currently on Ibrutinib therapy or whom have relapsed while on Ibrutinib treatment. Methods: Relapsed/refractory CLL patients (n=22) who were placed on Ibrutinib for progressive disease provided blood samples at a median of 3.2 months after Ibrutinib therapy initiation for these studies. We also obtained sequential samples on 8 patients from initial start of ibrutinib therapy and then over a 6 month follow-up period. CLL B-cells from these blood samples were subject to Ficoll separation, purified by using a Rosette Sep B-cell enrichment kit and then studied by flow cytometry to determine Axl RTK expression levels by flow cytometric analysis. Purified CLL B-cells (CD19+/CD5+) were cultured with TP-0903 in vitroat increasing doses (0.01µM - 0.50µM) for 24 hours and the LD50 dose was determined. In addition, 3 CLL patients who had been on Ibrutinib therapy and had a documented relapse were studied in similar fashion using TP-0903. LD50-sensitivity was measured. "LD50-sensitivity" was defined as an LD50 ≤0.50µM and "insensitive" was defined as an LD50 dose >0.50µM. CLL prognostic factors (e.g., FISH, IGHV mutation status, Rai stage, CD38, and CD49d) were evaluated at the time of ibrutinib treatment. Differences in factors between sensitive and insensitive cases were computed using the Kruskal-Wallis test for continuous variables and Chi-square test for categorical variables. Results: Twenty-two CLL patients (5 female, 17 male) were included in the analysis. Fourteen (64%) patients were found to be TP-0903 LD50-sensitive. Axl expression on CLL B-cells for this cohort was heterogeneous with a median of CD19+/CD5+ cells positive for Axl at 69.9% (range of 2.7-91.3%). The sensitive subjects tended to be younger with a median age at Ibrutinib treatment initiation of 62 vs 75.5 years (p=0.004). There were no significant differences in gender, FISH, IGHV mutation status, CD38, CD49d, or Rai stage between the sensitive and insensitive LD50 groups. There were no significant differences in relation to median Axl expression on CLL B-cells (sensitive: 72.6%, range: 2.7-91.3%; insensitive: 41.5%, range: 16.5-83.1%; p=0.35). The median number of treatments prior to initiation of ibrutinib did not differ between sensitivity groups (sensitive: 2.53, range: 8-10; insensitive: 43.5, range 12-20; p=0.2833). Association for ZAP70+ CLL B-cells tended to have more apoptosis induction by TP-0903 (sensitive: 84.6% ZAP70+; insensitive: 42.9% ZAP70+; p=0.052). In 8 CLL patients that were studied sequentially while on Ibrutinib continued to express Axl or increased their Axl expression (n=2) over a 3-6 month follow-up period. Three CLL patients who had relapsed on Ibrutinib were sensitive to TP-0903 with LD50 values of ≤0.50µM. Summary: Here we find that CLL B-cells from over 60% of relapsed CLL patients on Ibrutinib therapy were highly sensitive to the high-affinity Axl inhibitor TP-0903 with induction of apoptosis at nanomolar doses (≤0.50µM). The sensitivity of CLL B-cells to TP-0903 appears to be independent of Axl expression levels and of the known CLL prognostic factors but more evident for younger patients and for ZAP70+ expression status. Given this level of activity for apoptosis induction of CLL B-cells by TP-0903 encourages the further testing of this drug in clinical trials for CLL patients. Disclosures Parikh: Pharmacyclics: Honoraria, Research Funding. Shanafelt:Pharmacyclics: Research Funding; Janssen: Research Funding; Genentech: Research Funding; GlaxoSmithKline: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Hospira: Research Funding. Warner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Bearss:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Kay:Pharmacyclics: Research Funding; Tolero Pharmaceuticals: Research Funding; Acerta: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Morpho-Sys: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract Introduction: B-cell chronic lymphocytic leukemia (CLL) is an incurable disease despite that still requires options for treatment despite recent advances with BTK therapy. We previously found that Axl receptor tyrosine kinase (RTK), a member of the TAM (Tyro3, Axl, MER) family of RTKs, plays a critical role in CLL B-cell survival (Blood 2011, 117:1928). Here, we explored the levels of apoptosis induction of CLL B-cells using a novel high-affinity Axl inhibitor as a single agent or in combination with BTK inhibitors. Methods: Expression/activation status of the Tyro3, Axl and MER in CLL B-cells was evaluated by Immunoprecipitation/Western blots. Cells were treated with a high-affinity orally bioavailable Axl inhibitor TP-0903 (Tolero Pharmaceuticals) or FGFR inhibitor TKI-258 (Novartis) with or without the presence of CLL bone marrow stromal cells (BMSCs). Impact of Axl or FGFR signal inhibition on CLL B-cell survival was also evaluated by flow cytometry after staining with annexin V/propidium iodide. Finally, cells were exposed to TP-0903 in combination with BTK inhibitors to determine any synergistic/additive effects of the combination in induction of apoptosis. Combination index (CI) of two agents was calculated using the method of Chou and Talalay, 1984. Results: Although we defined pro-survival role of Axl in CLL B-cells, contribution of other members of the TAM family of RTKs including Tyro3 and MER to prolonged survival of the leukemic B-cells remains largely undefined. In this study we found that CLL B-cells overexpress Tyro3, but not MER, and that Tyro3 is constitutively phosphorylated. Interestingly, immunoprecipitation of Tyro3 from CLL B-cell lysates shows retention of highly active Axl in the immune complex suggesting that these two RTKs either heterodimerize or form a molecular complex in CLL B-cells. Our earlier work found that Axl regulates multiple cell survival signaling pathways including PI3K/AKT and Src and that, Axl inhibition using a first generation Axl inhibitor induced apoptosis in CLL B-cells (Blood 2011, 117:1928). Thus, we hypothesized that targeting Axl using a very high affinity Axl inhibitor would induce even higher levels of apoptosis in CLL B-cells compared to the first generation Axl inhibitor. To address this, we utilized a high-affinity orally bioavailable Axl inhibitor TP-0903 to generate preliminary information on its efficiency to specifically target Axl and the level of apoptosis induction in CLL B-cells. Indeed TP-0903 as a single agent induced a robust apoptotic cell death in CLL B-cells within 24 hours of exposure at nanomolar doses, which are easily achievable in vivo (information provided by Tolero Pharmaceuticals). Interestingly, while TP-0903 effectively reduces phosphorylation on Axl in CLL B-cells, it was unable to target P-Tyro3 suggesting that Axl is likely the predominant RTK of the TAM family in CLL B-cells. A marked reduction of the anti-apoptotic proteins Mcl-1, Bcl-2, XIAP and upregulation of the pro-apoptotic protein BIM in CLL B-cells via activation of its upstream regulator FOXO3a were noted as a result of inhibition of P-Axl and its downstream signaling pathways, P-AKT/P-Src. Importantly, TP-0903 was able to overcome CLL BMSC mediated protection of the leukemic B-cells from apoptosis induction. Given the observed robust induction of apoptosis with TP-0903 and the known impact of BTK inhibitors on CLL patient clinical outcome, we then exposed CLL B-cells to TP-0903 in combination with two BTK inhibitors that included Ibrutinib and a reversible BTK inhibitor TP-4216 (a kind gift from Tolero Pharmaceuticals). Based on the CI values, we found that the in vitro combination of TP-0903 and Ibrutinib showed additive effects in CLL B-cells from 6 of 11 CLL patients, CLL B-cells from the remaining 5 CLL patients showed antagonistic effects (Fig. 1A). However, we observed synergistic effects of TP-0903 in combination with TP-4216 in the induction of apoptosis in CLL B-cells from 2 of 10 patients and additive effects in CLL B-cells from 6 patients (Fig. 1B). Cells from the remaining 2 patients exhibited a slightly antagonisticeffect. Conclusion: In total, these results suggest that TP-0903 is quite effective in inducing robust CLL B-cell apoptosis as a single agent even at a nanomolar concentration and its combination with the reversible BTK inhibitor TP-4216 could be an effective and unique strategy for the treatment of CLL patients. Figure 1 Figure 1. Disclosures Warner: Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Bearss:Tolero Pharmaceuticals: Employment. Shanafelt:Genentech: Research Funding; GlaxoSmithKline: Research Funding; Celegene: Research Funding; Cephalon: Research Funding; Pharmacyclics/Jannsen: Research Funding; Hospira: Research Funding; Polyphenon E Int'l: Research Funding. Kay:Genetech: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

Perioperative serotonin syndrome has been associated with a number of medications and herbal supplements. We report a patient who developed serotonin syndrome immediately after an endoscopic procedure in which the preoperative use of black seed oil appears to have played a role in stimulating the syndrome. Black seed oil has not been previously reported in association with perioperative serotonin syndrome. Anesthesia professionals should be aware that patients taking black seed oil supplements may develop serotonin syndrome postoperatively.

Systemic mastocytosis (SM) is a rare clinical condition resulting from a clonal proliferation of abnormal mast cells. The clinical presentation may vary from mild cutaneous manifestations to aggressive systemic symptoms including intermittent episodes of anaphylaxis. We present a case of a 69-year-old male with abrupt and recurrent episodes of anaphylaxis with refractory distributive shock following cardiac surgery with cardiopulmonary bypass. Following a complex postoperative course, a bone marrow biopsy ultimately confirmed the diagnosis. Although rare, SM should be considered in the differential diagnosis of postoperative patients with unexplained and recurrent episodes of distributive shock.

AbstractIntroductionOur aim was to examine whether surgery with regional anesthesia (RA) is associated with accelerated long‐term cognitive decline comparable with that previously reported after general anesthesia (GA).MethodsLongitudinal cognitive function was analyzed in a cohort of 1819 older adults. Models assessed the rate of change in global and domain‐specific cognition over time in participants exposed to RA or GA.ResultsWhen compared with those unexposed to anesthesia, the postoperative rate of change of the cognitive global z‐score was greater in those exposed to both RA (difference in annual decline of −0.041, P = .011) and GA (−0.061, P < .001); these rates did not differ. In analysis of the domain‐specific scores, an accelerated decline in memory was observed after GA (−0.065, P < .001) but not RA (−0.011, P = .565).ConclusionsOlder adults undergoing surgery with RA experience decline of global cognition similar to those receiving GA; however, memory was not affected.

Objectives: Recurrent anterior glenohumeral instability is a disabling pathology that can be successfully treated by arthroscopic Bankart repair or an open Latarjet procedure. Long-term outcomes have shown lower rates of apprehension, recurrent dislocation and operative revision following Latarjet when compared to Bankart repair. However, there is a paucity of studies comparing the short-term post-operative recovery of arthroscopic Bankart versus open Latarjet. The purpose of this study is to evaluate the post-operative recovery following Bankart and the open Latarjet procedure. Methods: The surgical outcomes system (SOS) database (Arthrex Inc., Naples, FL) was used to compare the post-operative recovery outcomes after either a primary or revision arthroscopic Bankart and an open Latarjet procedure. Patients were included who had a minimum of 1 year follow-up. Preoperative and postoperative (2 weeks, 6 weeks, 3 months, 6 months, 1 year and 2 year) time points were evaluated. Outcomes measures included Visual Analog Pain Scale (VAS), American Shoulder and Elbow Surgeons (ASES) Shoulder Function Score, ASES Shoulder Index Score, and SANE Score. Overall, 787 patients underwent primary Bankart (518 male, 240 female, 4 not recorded), 36 underwent revision Bankart (24 male, 10 female, 2 not recorded) and 75 underwent an open Latarjet procedure (59 male, 12 female, 4 not recorded). The mean age for primary Bankart, revision Bankart, and open Latarjet was 40.8, 38.6 and 32.8 respectively. Additionally, the average BMI for primary Bankart, revision Bankart, and open Latarjet was 27.2, 28.13, and 25.6, respectively. Results: The postoperative recovery curves are displayed in Figure 1. When compared to primary Bankart, open Latarjet demonstrated significantly lower VAS scores at six weeks (p=0.0272) and at three months (p=0.0094). Medium term outcomes for ASES Shoulder Index Score, ASES Shoulder Function Score, and SANE Score, at 1- and 2-years showed no difference between primary Bankart and Latarjet. For the revision Bankart and open Latarjet procedures, the open Latarjet cohort demonstrated significantly higher ASES Shoulder Index Scores at the 3-months (p= 0.0017), 1-year (p= 0.0021), and 2-years (p= 0.0006) timepoints. Open Latarjet patients also had significantly higher ASES Shoulder Function Scores than revision Bankart at 3-months (p= 0.0162), 1-year (p= 0.0083), and 2-years (p= 0.0013). Revision Bankart repair resulted in significantly higher VAS scores than open Latarjet at 2-weeks (p= 0.0025), 6-weeks (p=0.0114), 3-months (p= 0.0024), 1-year (p= 0.0039), and 2-years (p= 0.0007). Conclusion: When compared to Bankart repair, open Latarjet provides improved pain and functional outcomes during the early recovery phase, 2-weeks, 6-weeks, 3-months, and 6-months, with equivalent medium-term outcomes at 1-2-years. Furthermore, when compared to revision Bankart reconstruction, open Latarjet provides improved ASES Shoulder Index Scores, ASES Shoulder Function Scores and VAS Scores at nearly all timepoints. In the treatment of recurrent anterior glenohumeral instability, open Latarjet is reasonable option in the primary setting and should be favored over Bankart repair for revision cases with improved pain relief and functional scores.

BACKGROUND: Intraoperative red blood cell (RBC) transfusion is common, yet transfusion strategies remain controversial as pretransfusion hemoglobin triggers are difficult to utilize during acute bleeding. Alternatively, postoperative hemoglobin values may provide useful information regarding transfusion practices, though optimal targets remain undefined. METHODS: This is a single-center observational cohort study of adults receiving allogeneic RBCs during noncardiac surgery from 2010 through 2014. Multivariable regression analyses adjusting for patient illness, laboratory derangements, and surgical features were used to assess relationships between initial postoperative hemoglobin values and a primary outcome of hospital-free days. RESULTS: A total of 8060 patients were included. Those with initial postoperative hemoglobin <7.5 or ≥11.5 g/dL had decreased hospital-free days [mean (95% confidence interval [CI]), −1.45 (−2.50 to −0.41) and −0.83 (−1.42 to −0.24), respectively] compared to a reference range of 9.5–10.4 g/dL (overall P value .003). For those with hemoglobin <7.5 g/dL, the odds (95% CI) for secondary outcomes included acute kidney injury (AKI) 1.43 (1.03–1.99), mortality 2.10 (1.18–3.74), and cerebral ischemia 3.12 (1.08–9.01). The odds for postoperative mechanical ventilation with hemoglobin ≥11.5 g/dL were 1.33 (1.07–1.65). Secondary outcome associations were not significant after multiple comparisons adjustment (Bonferroni P < .0056). CONCLUSIONS: In transfused patients, postoperative hemoglobin values between 7.5 and 11.5 g/dL were associated with superior outcomes compared to more extreme values. This range may represent a target for intraoperative transfusions, particularly during active bleeding when pretransfusion hemoglobin thresholds may be impractical or inaccurate. Given similar outcomes within this range, targeting hemoglobin at the lower aspect may be preferable, though prospective validation is warranted.

BACKGROUND: In January 2016, as part of the Maintenance of Certification in Anesthesiology (MOCA) program, the American Board of Anesthesiology launched MOCA Minute, a web-based longitudinal assessment, to supplant the former cognitive examination. We investigated the association between participation and performance in MOCA Minute and disciplinary actions against medical licenses of anesthesiologists. METHODS: All anesthesiologists with time-limited certificates (ie, certified in 2000 or after) who were required to register for MOCA Minute in 2016 were followed up through December 31, 2016. The incidence of postcertification prejudicial license actions was compared between those who did and did not register and compared between registrants who did and did not meet the MOCA Minute performance standard. RESULTS: The cumulative incidence of license actions was 1.2% (245/20,006) in anesthesiologists required to register for MOCA Minute. Nonregistration was associated with a higher incidence of license actions (hazard ratio, 2.93 [95% confidence interval {CI}, 2.15–4.00]). For the 18,534 (92.6%) who registered, later registration (after June 30, 2016) was associated with a higher incidence of license actions. In 2016, 16,308 (88.0%) anesthesiologists met the MOCA Minute performance standard. Of those not meeting the standard (n = 2226), most (n = 2093, 94.0%) failed because they did not complete the required 120 questions. Not meeting the standard was associated with a higher incidence of license actions (hazard ratio, 1.92 [95% CI, 1.36–2.72]). CONCLUSIONS: Both timely participation and meeting performance standard in MOCA Minute are associated with a lower likelihood of being disciplined by a state medical board.