Home | Molecular Cancer Therapeutics



Supplementary MaterialsSafe and Effective Sarcoma Therapy through Bispecific?Targeting of?EGFR and uPARAntonella Borgatti1,2,3, Joseph S. Koopmeiners3,4, Aaron L. Sarver3, Amber L. Winter5, Kathleen Stuebner5, Deborah Todhunter3,6, Anthony E. Rizzardi7, Jonathan C. Henriksen7, Stephen Schmechel7, Colleen L. Forster8, Jong-Hyuk Kim1,2,3, Jerry Froelich9, Jillian Walz1,2, Michael S. Henson1,2,3, Matthew Breen10,11, Kerstin Lindblad-Toh12,13, Felix Oh6, Kristy Pilbeam14, Jaime F. Modiano1,2,3,15,16, Daniel A. Vallera1,3,6Detailed Methods for ImmunohistochemistryImmunohistochemical staining for EGFR and uPAR in human and canine tissues was optimized for Aperio quantification as described [1,2]. Vimentin staining (Zymed cat# 18-0052 clone V9) mouse monoclonal (ThermoFisher) was used as a control for viable tissue; regions of negative staining were excluded from image analysis. CD31 staining (CD31 Dako, cat#M0823, Clone JC70A, ) was used to localize the regions of tumor used for analysis and quantification. Antibody was used at a 1:100 for dilution. Heat retrieval was done prior to staining in citrate buffer (pH 6) for 30 minutes with 20 minutes cool down prior to staining. The antibodies used to stain EGFR and uPAR, respectively, were anti-EGFR precursor antibody produced in rabbit (Sigma Prestige cat# HPA018530, ), and clone R4 mouse anti-uPAR monoclonal antibody (Dako cat # M7294, ). Methods for immunohistochemistry were the same for all antibodies.Detailed Description of the Canine Clinical StudyWe evaluated the safety and efficacy of adjuvant eBAT using a novel Bayesian adaptive Phase I-II trial design [3]. The primary objective of the design was to identify the biologically active (BA) dose based on a pre-defined criterion that considered the trade-off between safety and efficacy. Inference about safety and efficacy was based on the posterior distribution for the probability of 6-month survival and the probability of dose limiting toxicity (DLT) at each dose. Dose-finding was guided by a novel dose-response model that evaluated toxicity and efficacy as time-to-event outcomes, allowing new cohorts to be enrolled before previous cohorts have been fully observed and dramatically reducing trial duration. Dose-levels were considered acceptable if there was high posterior probability that the probability of toxicity was below a pre-defined threshold and the probability of 6-month survival was above a pre-defined threshold. Acceptable doses were ranked using a desirability index that considers a trade-off between efficacy and toxicity [4]. Dose-finding was completed in cohorts of three and dogs were treated at the dose that maximized the desirability index under the restriction that untried dose-levels could not be skipped during escalation. The dose that maximized the desirability index at trial completion was declared the BA dose.Owners of each dog gave written informed consent to treat prior to study entry. All dogs were treated at the University of Minnesota Veterinary Medical Center (VMC); the study was managed by the Clinical Investigation Center (CIC) of the College of Veterinary Medicine, University of Minnesota in compliance with principles of Good Clinical Practice [5].Inclusion criteria included histopathologically-confirmed diagnosis of stage-I (no evidence of tumor rupture) or stage-II (evidence of tumor rupture) HSA confined to the spleen; no evidence of regional or distant metastatic disease based on thoracic radiography and abdominal ultrasonography that was grossly confirmed at the time of surgery; no concurrent treatment with herbal treatments or supplements; performance score of 0 or 1 according to the Eastern Cooperative Oncology Group (ECOG) performance scale [6]; adequate organ function; no serious comorbidities, such as renal or hepatic failure, congestive heart failure, or clinical coagulopathy. Dogs were required to have a splenectomy prior to study entry. Each dog received a baseline complete history, physical examination, and pre-dose laboratory assessment that included a complete blood count (CBC), serum biochemical profile, coagulation parameters (PT/PTT) and urinalysis. Thoracic radiography and abdominal ultrasonography were performed prior to enrollment to rule out gross metastatic disease. eBAT was administered in a single cycle of three intravenous treatments at days 1, 3, and 5 at escalating doses of 25 ?g/kg/day (dose level 1), 50 ?g/kg/day (dose level 2), or 100 ?g/kg/day (dose level 3). Cohorts of three dogs were treated at each dose level and intra-patient dose escalations were not permitted. The protocol was modified starting with the fifth dog to include pre-loading with intravenous fluids at a rate of about 0.1 to 1 ml/kg/hr for 10 to 60 minutes. The drug was administered as a slow infusion over 5-20 minutes depending on volume and size of the dog. In total, the investigators were in contact with 181 families via email or telephone to assess their dog’s eligibility to participate in SRCBST-1. Of these, 79 dogs had surgery to remove a grossly abnormal spleen between November 28, 2012 and May 6, 2015 (51 at the VMC and 28 at another hospital prior to referral) and 23 dogs were enrolled in the study. One of these 23 dogs was euthanized at study Day 18 due to metastatic dissemination to the liver with rupture and hemoabdomen. This dog did not receive doxorubicin chemotherapy, but was included in all the analyses.Disease reassessment included physical examination, blood and urine evaluation, thoracic radiography and abdominal ultrasonography prior to doxorubicin treatments # 3 and # 5. No medications were prescribed or administered concurrently, unless needed to manage toxicity or other, unrelated medical conditions. Adverse events related to the study drug or to doxorubicin chemotherapy were treated with supportive care, as needed. Gastrointestinal toxicities were managed with famotidine, omeprazole, metronidazole, metoclopramide, ondansetron, and/or maropitant. Antibiotic therapy was allowed for prophylaxis in the event of severe neutropenia (counts <1,000/?l) or febrile neutropenia. Non-steroidal anti-inflammatory drugs or other analgesics (tramadol, gabapentin) were allowable for pain control as needed. Baseline characteristics for all dogs and by dose are summarized in Table 1A. The study protocol is summarized in Table 1B. The most common laboratory abnormalities at the time of screening included mild regenerative anemia (11 dogs), thrombocytosis (19 dogs), mild to moderate ALP elevation (six dogs), isosthenuria (four dogs), and slight ALT and AST elevation or slight hypoalbuminemia or proteinuria (one dog each). Slides were available for review by one of the study pathologists for 15 dogs. Most of the cases had mixed morphology, with areas showing capillary, cavernous, or solid organization. Mitotic indices also were comparable for each of these cases.A historical comparison group (Comparison group) consisted of 28 dogs with stage-I (8 dogs) or stage-II (20 dogs) HSA treated with SOC alone (surgery followed by adjuvant chemotherapy) at the VMC between 2005 and 2014. Chemotherapy treatment in this group consisted of metronomic piroxicam and cyclophosphamide in 2 dogs, doxorubicin chemotherapy in 8 dogs, and doxorubicin chemotherapy combined with metronomic cyclophosphamide in 20 dogs. Of these 20 dogs, one was switched to a CCNU/dacarbazine regimen due to progressive disease, and in another dog metronomic chlorambucil was used to replace cyclophosphamide following the development of sterile hemorrhagic cystitis. Survival times for all dogs were calculated from the date of diagnosis to the date of death.eBAT Pharmacokinetics and Neutralizing Antibody AssaysSerum samples were collected before starting treatment (time 0) and 5, 15, 30, 45, and 60 minutes after the end of the infusion on days 1 and 5 or 6 to measure drug pharmacokinetics (PK). Single serum samples were collected on days 8 and 21 to assess the presence of neutralizing antibodies (NAs). A bioassay with human RD cells was used to measure eBAT PK, as reported. Cells were incubated overnight prior to addition of the clinical batch eBAT as reported PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TY2hhcHBhPC9BdXRob3I+PFllYXI+MjAxMzwvWWVhcj48

UmVjTnVtPjk0PC9SZWNOdW0+PERpc3BsYXlUZXh0PlsyMl08L0Rpc3BsYXlUZXh0PjxyZWNvcmQ+

PHJlYy1udW1iZXI+OTQ8L3JlYy1udW1iZXI+PGZvcmVpZ24ta2V5cz48a2V5IGFwcD0iRU4iIGRi

LWlkPSJ0eGZ2djIydHh0cngwaGV4OWFyNXIyc2NwZDJ2cjIwdmYwdjUiIHRpbWVzdGFtcD0iMTQy

MjgxODY1NSI+OTQ8L2tleT48L2ZvcmVpZ24ta2V5cz48cmVmLXR5cGUgbmFtZT0iSm91cm5hbCBB

cnRpY2xlIj4xNzwvcmVmLXR5cGU+PGNvbnRyaWJ1dG9ycz48YXV0aG9ycz48YXV0aG9yPlNjaGFw

cGEsIEouIFQuPC9hdXRob3I+PGF1dGhvcj5GcmFudHosIEEuIE0uPC9hdXRob3I+PGF1dGhvcj5H

b3JkZW4sIEIuIEguPC9hdXRob3I+PGF1dGhvcj5EaWNrZXJzb24sIEUuIEIuPC9hdXRob3I+PGF1

dGhvcj5WYWxsZXJhLCBELiBBLjwvYXV0aG9yPjxhdXRob3I+TW9kaWFubywgSi4gRi48L2F1dGhv

cj48L2F1dGhvcnM+PC9jb250cmlidXRvcnM+PGF1dGgtYWRkcmVzcz5WZXRlcmluYXJ5IENsaW5p

Y2FsIFNjaWVuY2VzLCBVbml2ZXJzaXR5IG9mIE1pbm5lc290YSwgTWlubmVhcG9saXMsIE1OLCBV

U0EuIHNjaGEwNzc3QHVtbi5lZHU8L2F1dGgtYWRkcmVzcz48dGl0bGVzPjx0aXRsZT5IZW1hbmdp

b3NhcmNvbWEgYW5kIGl0cyBjYW5jZXIgc3RlbSBjZWxsIHN1YnBvcHVsYXRpb24gYXJlIGVmZmVj

dGl2ZWx5IGtpbGxlZCBieSBhIHRveGluIHRhcmdldGVkIHRocm91Z2ggZXBpZGVybWFsIGdyb3d0

aCBmYWN0b3IgYW5kIHVyb2tpbmFzZSByZWNlcHRvcnM8L3RpdGxlPjxzZWNvbmRhcnktdGl0bGU+

SW50IEogQ2FuY2VyPC9zZWNvbmRhcnktdGl0bGU+PGFsdC10aXRsZT5JbnRlcm5hdGlvbmFsIGpv

dXJuYWwgb2YgY2FuY2VyLiBKb3VybmFsIGludGVybmF0aW9uYWwgZHUgY2FuY2VyPC9hbHQtdGl0

bGU+PC90aXRsZXM+PHBlcmlvZGljYWw+PGZ1bGwtdGl0bGU+SW50IEogQ2FuY2VyPC9mdWxsLXRp

dGxlPjxhYmJyLTE+SW50ZXJuYXRpb25hbCBqb3VybmFsIG9mIGNhbmNlci4gSm91cm5hbCBpbnRl

cm5hdGlvbmFsIGR1IGNhbmNlcjwvYWJici0xPjwvcGVyaW9kaWNhbD48YWx0LXBlcmlvZGljYWw+

PGZ1bGwtdGl0bGU+SW50IEogQ2FuY2VyPC9mdWxsLXRpdGxlPjxhYmJyLTE+SW50ZXJuYXRpb25h

bCBqb3VybmFsIG9mIGNhbmNlci4gSm91cm5hbCBpbnRlcm5hdGlvbmFsIGR1IGNhbmNlcjwvYWJi

ci0xPjwvYWx0LXBlcmlvZGljYWw+PHBhZ2VzPjE5MzYtNDQ8L3BhZ2VzPjx2b2x1bWU+MTMzPC92

b2x1bWU+PG51bWJlcj44PC9udW1iZXI+PGVkaXRpb24+MjAxMy8wNC8wNTwvZWRpdGlvbj48a2V5

d29yZHM+PGtleXdvcmQ+QW5pbWFsczwva2V5d29yZD48a2V5d29yZD5BbnVyYTwva2V5d29yZD48

a2V5d29yZD5CYWN0ZXJpYWwgUHJvdGVpbnMvIHBoYXJtYWNvbG9neS90b3hpY2l0eTwva2V5d29y

ZD48a2V5d29yZD5DYXRzPC9rZXl3b3JkPjxrZXl3b3JkPkNlbGwgTGluZSwgVHVtb3I8L2tleXdv

cmQ+PGtleXdvcmQ+Q3l0b3RveGlucy9waGFybWFjb2xvZ3kvdG94aWNpdHk8L2tleXdvcmQ+PGtl

eXdvcmQ+RG9nczwva2V5d29yZD48a2V5d29yZD5IZW1hbmdpb3NhcmNvbWEvIGRydWcgdGhlcmFw

eTwva2V5d29yZD48a2V5d29yZD5IdW1hbnM8L2tleXdvcmQ+PGtleXdvcmQ+SW1tdW5vdG94aW5z

L3BoYXJtYWNvbG9neS90b3hpY2l0eTwva2V5d29yZD48a2V5d29yZD5OZW9wbGFzdGljIFN0ZW0g

Q2VsbHMvIGRydWcgZWZmZWN0czwva2V5d29yZD48a2V5d29yZD5SZWNlcHRvciwgRXBpZGVybWFs

IEdyb3d0aCBGYWN0b3IvIG1ldGFib2xpc208L2tleXdvcmQ+PGtleXdvcmQ+UmVjZXB0b3JzLCBV

cm9raW5hc2UgUGxhc21pbm9nZW4gQWN0aXZhdG9yLyBtZXRhYm9saXNtPC9rZXl3b3JkPjxrZXl3

b3JkPlJlY29tYmluYW50IEZ1c2lvbiBQcm90ZWlucy9waGFybWFjb2xvZ3kvdG94aWNpdHk8L2tl

eXdvcmQ+PC9rZXl3b3Jkcz48ZGF0ZXM+PHllYXI+MjAxMzwveWVhcj48cHViLWRhdGVzPjxkYXRl

Pk9jdCAxNTwvZGF0ZT48L3B1Yi1kYXRlcz48L2RhdGVzPjxpc2JuPjEwOTctMDIxNSAoRWxlY3Ry

b25pYykmI3hEOzAwMjAtNzEzNiAoTGlua2luZyk8L2lzYm4+PGFjY2Vzc2lvbi1udW0+MjM1NTMz

NzE8L2FjY2Vzc2lvbi1udW0+PHVybHM+PC91cmxzPjxjdXN0b20yPlBNQzM5ODUyNzU8L2N1c3Rv

bTI+PGN1c3RvbTY+TmlobXM1NzA0OTg8L2N1c3RvbTY+PGVsZWN0cm9uaWMtcmVzb3VyY2UtbnVt

PjEwLjEwMDIvaWpjLjI4MTg3PC9lbGVjdHJvbmljLXJlc291cmNlLW51bT48cmVtb3RlLWRhdGFi

YXNlLXByb3ZpZGVyPk5MTTwvcmVtb3RlLWRhdGFiYXNlLXByb3ZpZGVyPjxsYW5ndWFnZT5lbmc8

L2xhbmd1YWdlPjwvcmVjb3JkPjwvQ2l0ZT48L0VuZE5vdGU+

ADDIN EN.CITE PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TY2hhcHBhPC9BdXRob3I+PFllYXI+MjAxMzwvWWVhcj48

UmVjTnVtPjk0PC9SZWNOdW0+PERpc3BsYXlUZXh0PlsyMl08L0Rpc3BsYXlUZXh0PjxyZWNvcmQ+

PHJlYy1udW1iZXI+OTQ8L3JlYy1udW1iZXI+PGZvcmVpZ24ta2V5cz48a2V5IGFwcD0iRU4iIGRi

LWlkPSJ0eGZ2djIydHh0cngwaGV4OWFyNXIyc2NwZDJ2cjIwdmYwdjUiIHRpbWVzdGFtcD0iMTQy

MjgxODY1NSI+OTQ8L2tleT48L2ZvcmVpZ24ta2V5cz48cmVmLXR5cGUgbmFtZT0iSm91cm5hbCBB

cnRpY2xlIj4xNzwvcmVmLXR5cGU+PGNvbnRyaWJ1dG9ycz48YXV0aG9ycz48YXV0aG9yPlNjaGFw

cGEsIEouIFQuPC9hdXRob3I+PGF1dGhvcj5GcmFudHosIEEuIE0uPC9hdXRob3I+PGF1dGhvcj5H

b3JkZW4sIEIuIEguPC9hdXRob3I+PGF1dGhvcj5EaWNrZXJzb24sIEUuIEIuPC9hdXRob3I+PGF1

dGhvcj5WYWxsZXJhLCBELiBBLjwvYXV0aG9yPjxhdXRob3I+TW9kaWFubywgSi4gRi48L2F1dGhv

cj48L2F1dGhvcnM+PC9jb250cmlidXRvcnM+PGF1dGgtYWRkcmVzcz5WZXRlcmluYXJ5IENsaW5p

Y2FsIFNjaWVuY2VzLCBVbml2ZXJzaXR5IG9mIE1pbm5lc290YSwgTWlubmVhcG9saXMsIE1OLCBV

U0EuIHNjaGEwNzc3QHVtbi5lZHU8L2F1dGgtYWRkcmVzcz48dGl0bGVzPjx0aXRsZT5IZW1hbmdp

b3NhcmNvbWEgYW5kIGl0cyBjYW5jZXIgc3RlbSBjZWxsIHN1YnBvcHVsYXRpb24gYXJlIGVmZmVj

dGl2ZWx5IGtpbGxlZCBieSBhIHRveGluIHRhcmdldGVkIHRocm91Z2ggZXBpZGVybWFsIGdyb3d0

aCBmYWN0b3IgYW5kIHVyb2tpbmFzZSByZWNlcHRvcnM8L3RpdGxlPjxzZWNvbmRhcnktdGl0bGU+

SW50IEogQ2FuY2VyPC9zZWNvbmRhcnktdGl0bGU+PGFsdC10aXRsZT5JbnRlcm5hdGlvbmFsIGpv

dXJuYWwgb2YgY2FuY2VyLiBKb3VybmFsIGludGVybmF0aW9uYWwgZHUgY2FuY2VyPC9hbHQtdGl0

bGU+PC90aXRsZXM+PHBlcmlvZGljYWw+PGZ1bGwtdGl0bGU+SW50IEogQ2FuY2VyPC9mdWxsLXRp

dGxlPjxhYmJyLTE+SW50ZXJuYXRpb25hbCBqb3VybmFsIG9mIGNhbmNlci4gSm91cm5hbCBpbnRl

cm5hdGlvbmFsIGR1IGNhbmNlcjwvYWJici0xPjwvcGVyaW9kaWNhbD48YWx0LXBlcmlvZGljYWw+

PGZ1bGwtdGl0bGU+SW50IEogQ2FuY2VyPC9mdWxsLXRpdGxlPjxhYmJyLTE+SW50ZXJuYXRpb25h

bCBqb3VybmFsIG9mIGNhbmNlci4gSm91cm5hbCBpbnRlcm5hdGlvbmFsIGR1IGNhbmNlcjwvYWJi

ci0xPjwvYWx0LXBlcmlvZGljYWw+PHBhZ2VzPjE5MzYtNDQ8L3BhZ2VzPjx2b2x1bWU+MTMzPC92

b2x1bWU+PG51bWJlcj44PC9udW1iZXI+PGVkaXRpb24+MjAxMy8wNC8wNTwvZWRpdGlvbj48a2V5

d29yZHM+PGtleXdvcmQ+QW5pbWFsczwva2V5d29yZD48a2V5d29yZD5BbnVyYTwva2V5d29yZD48

a2V5d29yZD5CYWN0ZXJpYWwgUHJvdGVpbnMvIHBoYXJtYWNvbG9neS90b3hpY2l0eTwva2V5d29y

ZD48a2V5d29yZD5DYXRzPC9rZXl3b3JkPjxrZXl3b3JkPkNlbGwgTGluZSwgVHVtb3I8L2tleXdv

cmQ+PGtleXdvcmQ+Q3l0b3RveGlucy9waGFybWFjb2xvZ3kvdG94aWNpdHk8L2tleXdvcmQ+PGtl

eXdvcmQ+RG9nczwva2V5d29yZD48a2V5d29yZD5IZW1hbmdpb3NhcmNvbWEvIGRydWcgdGhlcmFw

eTwva2V5d29yZD48a2V5d29yZD5IdW1hbnM8L2tleXdvcmQ+PGtleXdvcmQ+SW1tdW5vdG94aW5z

L3BoYXJtYWNvbG9neS90b3hpY2l0eTwva2V5d29yZD48a2V5d29yZD5OZW9wbGFzdGljIFN0ZW0g

Q2VsbHMvIGRydWcgZWZmZWN0czwva2V5d29yZD48a2V5d29yZD5SZWNlcHRvciwgRXBpZGVybWFs

IEdyb3d0aCBGYWN0b3IvIG1ldGFib2xpc208L2tleXdvcmQ+PGtleXdvcmQ+UmVjZXB0b3JzLCBV

cm9raW5hc2UgUGxhc21pbm9nZW4gQWN0aXZhdG9yLyBtZXRhYm9saXNtPC9rZXl3b3JkPjxrZXl3

b3JkPlJlY29tYmluYW50IEZ1c2lvbiBQcm90ZWlucy9waGFybWFjb2xvZ3kvdG94aWNpdHk8L2tl

eXdvcmQ+PC9rZXl3b3Jkcz48ZGF0ZXM+PHllYXI+MjAxMzwveWVhcj48cHViLWRhdGVzPjxkYXRl

Pk9jdCAxNTwvZGF0ZT48L3B1Yi1kYXRlcz48L2RhdGVzPjxpc2JuPjEwOTctMDIxNSAoRWxlY3Ry

b25pYykmI3hEOzAwMjAtNzEzNiAoTGlua2luZyk8L2lzYm4+PGFjY2Vzc2lvbi1udW0+MjM1NTMz

NzE8L2FjY2Vzc2lvbi1udW0+PHVybHM+PC91cmxzPjxjdXN0b20yPlBNQzM5ODUyNzU8L2N1c3Rv

bTI+PGN1c3RvbTY+TmlobXM1NzA0OTg8L2N1c3RvbTY+PGVsZWN0cm9uaWMtcmVzb3VyY2UtbnVt

PjEwLjEwMDIvaWpjLjI4MTg3PC9lbGVjdHJvbmljLXJlc291cmNlLW51bT48cmVtb3RlLWRhdGFi

YXNlLXByb3ZpZGVyPk5MTTwvcmVtb3RlLWRhdGFiYXNlLXByb3ZpZGVyPjxsYW5ndWFnZT5lbmc8

L2xhbmd1YWdlPjwvcmVjb3JkPjwvQ2l0ZT48L0VuZE5vdGU+

ADDIN EN.CITE.DATA [7]. Proliferation was measured after 72 hours using a standard thymidine uptake assay. The presence of eBAT in serum was extrapolated from the standard curve as reported PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5LcmVpdG1hbjwvQXV0aG9yPjxZZWFyPjIwMTI8L1llYXI+

PFJlY051bT4xNzQ8L1JlY051bT48RGlzcGxheVRleHQ+WzM2XTwvRGlzcGxheVRleHQ+PHJlY29y

ZD48cmVjLW51bWJlcj4xNzQ8L3JlYy1udW1iZXI+PGZvcmVpZ24ta2V5cz48a2V5IGFwcD0iRU4i

IGRiLWlkPSJ0eGZ2djIydHh0cngwaGV4OWFyNXIyc2NwZDJ2cjIwdmYwdjUiIHRpbWVzdGFtcD0i

MTQ2Mzk1MTI4NyI+MTc0PC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10eXBlIG5hbWU9IkpvdXJu

YWwgQXJ0aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+PGF1dGhvcnM+PGF1dGhvcj5L

cmVpdG1hbiwgUi4gSi48L2F1dGhvcj48YXV0aG9yPlRhbGxtYW4sIE0uIFMuPC9hdXRob3I+PGF1

dGhvcj5Sb2JhaywgVC48L2F1dGhvcj48YXV0aG9yPkNvdXRyZSwgUy48L2F1dGhvcj48YXV0aG9y

PldpbHNvbiwgVy4gSC48L2F1dGhvcj48YXV0aG9yPlN0ZXRsZXItU3RldmVuc29uLCBNLjwvYXV0

aG9yPjxhdXRob3I+Rml0emdlcmFsZCwgRC4gSi48L2F1dGhvcj48YXV0aG9yPkxlY2hsZWlkZXIs

IFIuPC9hdXRob3I+PGF1dGhvcj5QYXN0YW4sIEkuPC9hdXRob3I+PC9hdXRob3JzPjwvY29udHJp

YnV0b3JzPjxhdXRoLWFkZHJlc3M+TmF0aW9uYWwgQ2FuY2VyIEluc3RpdHV0ZSwgTmF0aW9uYWwg

SW5zdGl0dXRlc29mIEhlYWx0aCwgQmV0aGVzZGEsIFVTQS4ga3JlaXRtYXJAbWFpbC5uaWguZ292

PC9hdXRoLWFkZHJlc3M+PHRpdGxlcz48dGl0bGU+UGhhc2UgSSB0cmlhbCBvZiBhbnRpLUNEMjIg

cmVjb21iaW5hbnQgaW1tdW5vdG94aW4gbW94ZXR1bW9tYWIgcGFzdWRvdG94IChDQVQtODAxNSBv

ciBIQTIyKSBpbiBwYXRpZW50cyB3aXRoIGhhaXJ5IGNlbGwgbGV1a2VtaWE8L3RpdGxlPjxzZWNv

bmRhcnktdGl0bGU+SiBDbGluIE9uY29sPC9zZWNvbmRhcnktdGl0bGU+PC90aXRsZXM+PHBlcmlv

ZGljYWw+PGZ1bGwtdGl0bGU+SiBDbGluIE9uY29sPC9mdWxsLXRpdGxlPjwvcGVyaW9kaWNhbD48

cGFnZXM+MTgyMi04PC9wYWdlcz48dm9sdW1lPjMwPC92b2x1bWU+PG51bWJlcj4xNTwvbnVtYmVy

PjxrZXl3b3Jkcz48a2V5d29yZD5BZHVsdDwva2V5d29yZD48a2V5d29yZD5BZ2VkPC9rZXl3b3Jk

PjxrZXl3b3JkPkJhY3RlcmlhbCBUb3hpbnMvYWR2ZXJzZSBlZmZlY3RzLyp0aGVyYXBldXRpYyB1

c2U8L2tleXdvcmQ+PGtleXdvcmQ+RGlzZWFzZS1GcmVlIFN1cnZpdmFsPC9rZXl3b3JkPjxrZXl3

b3JkPkRydWcgUmVzaXN0YW5jZSwgTmVvcGxhc208L2tleXdvcmQ+PGtleXdvcmQ+RXhvdG94aW5z

L2FkdmVyc2UgZWZmZWN0cy8qdGhlcmFwZXV0aWMgdXNlPC9rZXl3b3JkPjxrZXl3b3JkPkZlbWFs

ZTwva2V5d29yZD48a2V5d29yZD5IdW1hbnM8L2tleXdvcmQ+PGtleXdvcmQ+SW1tdW5vdGhlcmFw

eS9hZHZlcnNlIGVmZmVjdHMvKm1ldGhvZHM8L2tleXdvcmQ+PGtleXdvcmQ+TGV1a2VtaWEsIEhh

aXJ5IENlbGwvaW1tdW5vbG9neS9wYXRob2xvZ3kvKnRoZXJhcHk8L2tleXdvcmQ+PGtleXdvcmQ+

TWFsZTwva2V5d29yZD48a2V5d29yZD5NaWRkbGUgQWdlZDwva2V5d29yZD48a2V5d29yZD5TaWFs

aWMgQWNpZCBCaW5kaW5nIElnLWxpa2UgTGVjdGluIDIvKmltbXVub2xvZ3k8L2tleXdvcmQ+PGtl

eXdvcmQ+VGltZSBGYWN0b3JzPC9rZXl3b3JkPjxrZXl3b3JkPlRyZWF0bWVudCBPdXRjb21lPC9r

ZXl3b3JkPjwva2V5d29yZHM+PGRhdGVzPjx5ZWFyPjIwMTI8L3llYXI+PHB1Yi1kYXRlcz48ZGF0

ZT5NYXkgMjA8L2RhdGU+PC9wdWItZGF0ZXM+PC9kYXRlcz48aXNibj4xNTI3LTc3NTUgKEVsZWN0

cm9uaWMpJiN4RDswNzMyLTE4M1ggKExpbmtpbmcpPC9pc2JuPjxhY2Nlc3Npb24tbnVtPjIyMzU1

MDUzPC9hY2Nlc3Npb24tbnVtPjx1cmxzPjxyZWxhdGVkLXVybHM+PHVybD5odHRwOi8vd3d3Lm5j

YmkubmxtLm5paC5nb3YvcHVibWVkLzIyMzU1MDUzPC91cmw+PC9yZWxhdGVkLXVybHM+PC91cmxz

PjxjdXN0b20yPlBNQzMzODMxODE8L2N1c3RvbTI+PGVsZWN0cm9uaWMtcmVzb3VyY2UtbnVtPjEw

LjEyMDAvSkNPLjIwMTEuMzguMTc1NjwvZWxlY3Ryb25pYy1yZXNvdXJjZS1udW0+PC9yZWNvcmQ+

PC9DaXRlPjwvRW5kTm90ZT5=

ADDIN EN.CITE PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5LcmVpdG1hbjwvQXV0aG9yPjxZZWFyPjIwMTI8L1llYXI+

PFJlY051bT4xNzQ8L1JlY051bT48RGlzcGxheVRleHQ+WzM2XTwvRGlzcGxheVRleHQ+PHJlY29y

ZD48cmVjLW51bWJlcj4xNzQ8L3JlYy1udW1iZXI+PGZvcmVpZ24ta2V5cz48a2V5IGFwcD0iRU4i

IGRiLWlkPSJ0eGZ2djIydHh0cngwaGV4OWFyNXIyc2NwZDJ2cjIwdmYwdjUiIHRpbWVzdGFtcD0i

MTQ2Mzk1MTI4NyI+MTc0PC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10eXBlIG5hbWU9IkpvdXJu

YWwgQXJ0aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+PGF1dGhvcnM+PGF1dGhvcj5L

cmVpdG1hbiwgUi4gSi48L2F1dGhvcj48YXV0aG9yPlRhbGxtYW4sIE0uIFMuPC9hdXRob3I+PGF1

dGhvcj5Sb2JhaywgVC48L2F1dGhvcj48YXV0aG9yPkNvdXRyZSwgUy48L2F1dGhvcj48YXV0aG9y

PldpbHNvbiwgVy4gSC48L2F1dGhvcj48YXV0aG9yPlN0ZXRsZXItU3RldmVuc29uLCBNLjwvYXV0

aG9yPjxhdXRob3I+Rml0emdlcmFsZCwgRC4gSi48L2F1dGhvcj48YXV0aG9yPkxlY2hsZWlkZXIs

IFIuPC9hdXRob3I+PGF1dGhvcj5QYXN0YW4sIEkuPC9hdXRob3I+PC9hdXRob3JzPjwvY29udHJp

YnV0b3JzPjxhdXRoLWFkZHJlc3M+TmF0aW9uYWwgQ2FuY2VyIEluc3RpdHV0ZSwgTmF0aW9uYWwg

SW5zdGl0dXRlc29mIEhlYWx0aCwgQmV0aGVzZGEsIFVTQS4ga3JlaXRtYXJAbWFpbC5uaWguZ292

PC9hdXRoLWFkZHJlc3M+PHRpdGxlcz48dGl0bGU+UGhhc2UgSSB0cmlhbCBvZiBhbnRpLUNEMjIg

cmVjb21iaW5hbnQgaW1tdW5vdG94aW4gbW94ZXR1bW9tYWIgcGFzdWRvdG94IChDQVQtODAxNSBv

ciBIQTIyKSBpbiBwYXRpZW50cyB3aXRoIGhhaXJ5IGNlbGwgbGV1a2VtaWE8L3RpdGxlPjxzZWNv

bmRhcnktdGl0bGU+SiBDbGluIE9uY29sPC9zZWNvbmRhcnktdGl0bGU+PC90aXRsZXM+PHBlcmlv

ZGljYWw+PGZ1bGwtdGl0bGU+SiBDbGluIE9uY29sPC9mdWxsLXRpdGxlPjwvcGVyaW9kaWNhbD48

cGFnZXM+MTgyMi04PC9wYWdlcz48dm9sdW1lPjMwPC92b2x1bWU+PG51bWJlcj4xNTwvbnVtYmVy

PjxrZXl3b3Jkcz48a2V5d29yZD5BZHVsdDwva2V5d29yZD48a2V5d29yZD5BZ2VkPC9rZXl3b3Jk

PjxrZXl3b3JkPkJhY3RlcmlhbCBUb3hpbnMvYWR2ZXJzZSBlZmZlY3RzLyp0aGVyYXBldXRpYyB1

c2U8L2tleXdvcmQ+PGtleXdvcmQ+RGlzZWFzZS1GcmVlIFN1cnZpdmFsPC9rZXl3b3JkPjxrZXl3

b3JkPkRydWcgUmVzaXN0YW5jZSwgTmVvcGxhc208L2tleXdvcmQ+PGtleXdvcmQ+RXhvdG94aW5z

L2FkdmVyc2UgZWZmZWN0cy8qdGhlcmFwZXV0aWMgdXNlPC9rZXl3b3JkPjxrZXl3b3JkPkZlbWFs

ZTwva2V5d29yZD48a2V5d29yZD5IdW1hbnM8L2tleXdvcmQ+PGtleXdvcmQ+SW1tdW5vdGhlcmFw

eS9hZHZlcnNlIGVmZmVjdHMvKm1ldGhvZHM8L2tleXdvcmQ+PGtleXdvcmQ+TGV1a2VtaWEsIEhh

aXJ5IENlbGwvaW1tdW5vbG9neS9wYXRob2xvZ3kvKnRoZXJhcHk8L2tleXdvcmQ+PGtleXdvcmQ+

TWFsZTwva2V5d29yZD48a2V5d29yZD5NaWRkbGUgQWdlZDwva2V5d29yZD48a2V5d29yZD5TaWFs

aWMgQWNpZCBCaW5kaW5nIElnLWxpa2UgTGVjdGluIDIvKmltbXVub2xvZ3k8L2tleXdvcmQ+PGtl

eXdvcmQ+VGltZSBGYWN0b3JzPC9rZXl3b3JkPjxrZXl3b3JkPlRyZWF0bWVudCBPdXRjb21lPC9r

ZXl3b3JkPjwva2V5d29yZHM+PGRhdGVzPjx5ZWFyPjIwMTI8L3llYXI+PHB1Yi1kYXRlcz48ZGF0

ZT5NYXkgMjA8L2RhdGU+PC9wdWItZGF0ZXM+PC9kYXRlcz48aXNibj4xNTI3LTc3NTUgKEVsZWN0

cm9uaWMpJiN4RDswNzMyLTE4M1ggKExpbmtpbmcpPC9pc2JuPjxhY2Nlc3Npb24tbnVtPjIyMzU1

MDUzPC9hY2Nlc3Npb24tbnVtPjx1cmxzPjxyZWxhdGVkLXVybHM+PHVybD5odHRwOi8vd3d3Lm5j

YmkubmxtLm5paC5nb3YvcHVibWVkLzIyMzU1MDUzPC91cmw+PC9yZWxhdGVkLXVybHM+PC91cmxz

PjxjdXN0b20yPlBNQzMzODMxODE8L2N1c3RvbTI+PGVsZWN0cm9uaWMtcmVzb3VyY2UtbnVtPjEw

LjEyMDAvSkNPLjIwMTEuMzguMTc1NjwvZWxlY3Ryb25pYy1yZXNvdXJjZS1udW0+PC9yZWNvcmQ+

PC9DaXRlPjwvRW5kTm90ZT5=

ADDIN EN.CITE.DATA [8]. The presence of NAs was inferred from the capacity of serum samples to block cell death caused by reference eBAT PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5WYWxsZXJhPC9BdXRob3I+PFllYXI+MjAwNTwvWWVhcj48

UmVjTnVtPjE3NTwvUmVjTnVtPjxEaXNwbGF5VGV4dD5bMzddPC9EaXNwbGF5VGV4dD48cmVjb3Jk

PjxyZWMtbnVtYmVyPjE3NTwvcmVjLW51bWJlcj48Zm9yZWlnbi1rZXlzPjxrZXkgYXBwPSJFTiIg

ZGItaWQ9InR4ZnZ2MjJ0eHRyeDBoZXg5YXI1cjJzY3BkMnZyMjB2ZjB2NSIgdGltZXN0YW1wPSIx

NDYzOTUxMzIwIj4xNzU8L2tleT48L2ZvcmVpZ24ta2V5cz48cmVmLXR5cGUgbmFtZT0iSm91cm5h

bCBBcnRpY2xlIj4xNzwvcmVmLXR5cGU+PGNvbnRyaWJ1dG9ycz48YXV0aG9ycz48YXV0aG9yPlZh

bGxlcmEsIEQuIEEuPC9hdXRob3I+PGF1dGhvcj5Ub2RodW50ZXIsIEQuIEEuPC9hdXRob3I+PGF1

dGhvcj5LdXJva2ksIEQuIFcuPC9hdXRob3I+PGF1dGhvcj5TaHUsIFkuPC9hdXRob3I+PGF1dGhv

cj5TaWNoZW5lZGVyLCBBLjwvYXV0aG9yPjxhdXRob3I+Q2hlbiwgSC48L2F1dGhvcj48L2F1dGhv

cnM+PC9jb250cmlidXRvcnM+PGF1dGgtYWRkcmVzcz5EZXBhcnRtZW50IG9mIFRoZXJhcGV1dGlj

IFJhZGlvbG9neS1SYWRpYXRpb24gT25jb2xvZ3ksIFNlY3Rpb24gb24gTW9sZWN1bGFyIENhbmNl

ciBUaGVyYXBldXRpY3MsIFVuaXZlcnNpdHkgb2YgTWlubmVzb3RhIENhbmNlciBDZW50ZXIsIE1p

bm5lYXBvbGlzLCBNaW5uZXNvdGEsIFVTQS4gdmFsbGUwMDFAdW1uLmVkdTwvYXV0aC1hZGRyZXNz

Pjx0aXRsZXM+PHRpdGxlPkEgYmlzcGVjaWZpYyByZWNvbWJpbmFudCBpbW11bm90b3hpbiwgRFQy

MjE5LCB0YXJnZXRpbmcgaHVtYW4gQ0QxOSBhbmQgQ0QyMiByZWNlcHRvcnMgaW4gYSBtb3VzZSB4

ZW5vZ3JhZnQgbW9kZWwgb2YgQi1jZWxsIGxldWtlbWlhL2x5bXBob21hPC90aXRsZT48c2Vjb25k

YXJ5LXRpdGxlPkNsaW4gQ2FuY2VyIFJlczwvc2Vjb25kYXJ5LXRpdGxlPjwvdGl0bGVzPjxwZXJp

b2RpY2FsPjxmdWxsLXRpdGxlPkNsaW4gQ2FuY2VyIFJlczwvZnVsbC10aXRsZT48YWJici0xPkNs

aW5pY2FsIGNhbmNlciByZXNlYXJjaCA6IGFuIG9mZmljaWFsIGpvdXJuYWwgb2YgdGhlIEFtZXJp

Y2FuIEFzc29jaWF0aW9uIGZvciBDYW5jZXIgUmVzZWFyY2g8L2FiYnItMT48L3BlcmlvZGljYWw+

PHBhZ2VzPjM4NzktODg8L3BhZ2VzPjx2b2x1bWU+MTE8L3ZvbHVtZT48bnVtYmVyPjEwPC9udW1i

ZXI+PGtleXdvcmRzPjxrZXl3b3JkPkFuaW1hbHM8L2tleXdvcmQ+PGtleXdvcmQ+QW50aWJvZGll

czwva2V5d29yZD48a2V5d29yZD5BbnRpZ2VucywgQ0QvKmltbXVub2xvZ3k8L2tleXdvcmQ+PGtl

eXdvcmQ+QW50aWdlbnMsIENEMTkvKmltbXVub2xvZ3k8L2tleXdvcmQ+PGtleXdvcmQ+QW50aWdl

bnMsIERpZmZlcmVudGlhdGlvbiwgQi1MeW1waG9jeXRlLyppbW11bm9sb2d5PC9rZXl3b3JkPjxr

ZXl3b3JkPkNlbGwgQWRoZXNpb24gTW9sZWN1bGVzLyppbW11bm9sb2d5PC9rZXl3b3JkPjxrZXl3

b3JkPkNlbGwgRGVhdGg8L2tleXdvcmQ+PGtleXdvcmQ+RGlwaHRoZXJpYSBUb3hpbi9pbW11bm9s

b2d5PC9rZXl3b3JkPjxrZXl3b3JkPkVzY2hlcmljaGlhIGNvbGkvZ2VuZXRpY3MvaW1tdW5vbG9n

eTwva2V5d29yZD48a2V5d29yZD5GZW1hbGU8L2tleXdvcmQ+PGtleXdvcmQ+SW1tdW5vdG94aW5z

LyppbW11bm9sb2d5LypwaGFybWFjb2xvZ3k8L2tleXdvcmQ+PGtleXdvcmQ+TGVjdGlucy8qaW1t

dW5vbG9neTwva2V5d29yZD48a2V5d29yZD5MZXVrZW1pYSwgQi1DZWxsPC9rZXl3b3JkPjxrZXl3

b3JkPkx5bXBob21hLCBCLUNlbGw8L2tleXdvcmQ+PGtleXdvcmQ+TWljZTwva2V5d29yZD48a2V5

d29yZD5NaWNlLCBOdWRlPC9rZXl3b3JkPjxrZXl3b3JkPk1pY2UsIFNDSUQ8L2tleXdvcmQ+PGtl

eXdvcmQ+TW9sZWN1bGFyIENvbmZvcm1hdGlvbjwva2V5d29yZD48a2V5d29yZD5TaWFsaWMgQWNp

ZCBCaW5kaW5nIElnLWxpa2UgTGVjdGluIDI8L2tleXdvcmQ+PGtleXdvcmQ+U3Vydml2YWwgQW5h

bHlzaXM8L2tleXdvcmQ+PGtleXdvcmQ+VHJhbnNwbGFudGF0aW9uLCBIZXRlcm9sb2dvdXM8L2tl

eXdvcmQ+PGtleXdvcmQ+VHVtb3IgQ2VsbHMsIEN1bHR1cmVkPC9rZXl3b3JkPjwva2V5d29yZHM+

PGRhdGVzPjx5ZWFyPjIwMDU8L3llYXI+PHB1Yi1kYXRlcz48ZGF0ZT5NYXkgMTU8L2RhdGU+PC9w

dWItZGF0ZXM+PC9kYXRlcz48aXNibj4xMDc4LTA0MzIgKFByaW50KSYjeEQ7MTA3OC0wNDMyIChM

aW5raW5nKTwvaXNibj48YWNjZXNzaW9uLW51bT4xNTg5NzU4OTwvYWNjZXNzaW9uLW51bT48dXJs

cz48cmVsYXRlZC11cmxzPjx1cmw+aHR0cDovL3d3dy5uY2JpLm5sbS5uaWguZ292L3B1Ym1lZC8x

NTg5NzU4OTwvdXJsPjwvcmVsYXRlZC11cmxzPjwvdXJscz48ZWxlY3Ryb25pYy1yZXNvdXJjZS1u

dW0+MTAuMTE1OC8xMDc4LTA0MzIuQ0NSLTA0LTIyOTA8L2VsZWN0cm9uaWMtcmVzb3VyY2UtbnVt

PjwvcmVjb3JkPjwvQ2l0ZT48L0VuZE5vdGU+

ADDIN EN.CITE PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5WYWxsZXJhPC9BdXRob3I+PFllYXI+MjAwNTwvWWVhcj48

UmVjTnVtPjE3NTwvUmVjTnVtPjxEaXNwbGF5VGV4dD5bMzddPC9EaXNwbGF5VGV4dD48cmVjb3Jk

PjxyZWMtbnVtYmVyPjE3NTwvcmVjLW51bWJlcj48Zm9yZWlnbi1rZXlzPjxrZXkgYXBwPSJFTiIg

ZGItaWQ9InR4ZnZ2MjJ0eHRyeDBoZXg5YXI1cjJzY3BkMnZyMjB2ZjB2NSIgdGltZXN0YW1wPSIx

NDYzOTUxMzIwIj4xNzU8L2tleT48L2ZvcmVpZ24ta2V5cz48cmVmLXR5cGUgbmFtZT0iSm91cm5h

bCBBcnRpY2xlIj4xNzwvcmVmLXR5cGU+PGNvbnRyaWJ1dG9ycz48YXV0aG9ycz48YXV0aG9yPlZh

bGxlcmEsIEQuIEEuPC9hdXRob3I+PGF1dGhvcj5Ub2RodW50ZXIsIEQuIEEuPC9hdXRob3I+PGF1

dGhvcj5LdXJva2ksIEQuIFcuPC9hdXRob3I+PGF1dGhvcj5TaHUsIFkuPC9hdXRob3I+PGF1dGhv

cj5TaWNoZW5lZGVyLCBBLjwvYXV0aG9yPjxhdXRob3I+Q2hlbiwgSC48L2F1dGhvcj48L2F1dGhv

cnM+PC9jb250cmlidXRvcnM+PGF1dGgtYWRkcmVzcz5EZXBhcnRtZW50IG9mIFRoZXJhcGV1dGlj

IFJhZGlvbG9neS1SYWRpYXRpb24gT25jb2xvZ3ksIFNlY3Rpb24gb24gTW9sZWN1bGFyIENhbmNl

ciBUaGVyYXBldXRpY3MsIFVuaXZlcnNpdHkgb2YgTWlubmVzb3RhIENhbmNlciBDZW50ZXIsIE1p

bm5lYXBvbGlzLCBNaW5uZXNvdGEsIFVTQS4gdmFsbGUwMDFAdW1uLmVkdTwvYXV0aC1hZGRyZXNz

Pjx0aXRsZXM+PHRpdGxlPkEgYmlzcGVjaWZpYyByZWNvbWJpbmFudCBpbW11bm90b3hpbiwgRFQy

MjE5LCB0YXJnZXRpbmcgaHVtYW4gQ0QxOSBhbmQgQ0QyMiByZWNlcHRvcnMgaW4gYSBtb3VzZSB4

ZW5vZ3JhZnQgbW9kZWwgb2YgQi1jZWxsIGxldWtlbWlhL2x5bXBob21hPC90aXRsZT48c2Vjb25k

YXJ5LXRpdGxlPkNsaW4gQ2FuY2VyIFJlczwvc2Vjb25kYXJ5LXRpdGxlPjwvdGl0bGVzPjxwZXJp

b2RpY2FsPjxmdWxsLXRpdGxlPkNsaW4gQ2FuY2VyIFJlczwvZnVsbC10aXRsZT48YWJici0xPkNs

aW5pY2FsIGNhbmNlciByZXNlYXJjaCA6IGFuIG9mZmljaWFsIGpvdXJuYWwgb2YgdGhlIEFtZXJp

Y2FuIEFzc29jaWF0aW9uIGZvciBDYW5jZXIgUmVzZWFyY2g8L2FiYnItMT48L3BlcmlvZGljYWw+

PHBhZ2VzPjM4NzktODg8L3BhZ2VzPjx2b2x1bWU+MTE8L3ZvbHVtZT48bnVtYmVyPjEwPC9udW1i

ZXI+PGtleXdvcmRzPjxrZXl3b3JkPkFuaW1hbHM8L2tleXdvcmQ+PGtleXdvcmQ+QW50aWJvZGll

czwva2V5d29yZD48a2V5d29yZD5BbnRpZ2VucywgQ0QvKmltbXVub2xvZ3k8L2tleXdvcmQ+PGtl

eXdvcmQ+QW50aWdlbnMsIENEMTkvKmltbXVub2xvZ3k8L2tleXdvcmQ+PGtleXdvcmQ+QW50aWdl

bnMsIERpZmZlcmVudGlhdGlvbiwgQi1MeW1waG9jeXRlLyppbW11bm9sb2d5PC9rZXl3b3JkPjxr

ZXl3b3JkPkNlbGwgQWRoZXNpb24gTW9sZWN1bGVzLyppbW11bm9sb2d5PC9rZXl3b3JkPjxrZXl3

b3JkPkNlbGwgRGVhdGg8L2tleXdvcmQ+PGtleXdvcmQ+RGlwaHRoZXJpYSBUb3hpbi9pbW11bm9s

b2d5PC9rZXl3b3JkPjxrZXl3b3JkPkVzY2hlcmljaGlhIGNvbGkvZ2VuZXRpY3MvaW1tdW5vbG9n

eTwva2V5d29yZD48a2V5d29yZD5GZW1hbGU8L2tleXdvcmQ+PGtleXdvcmQ+SW1tdW5vdG94aW5z

LyppbW11bm9sb2d5LypwaGFybWFjb2xvZ3k8L2tleXdvcmQ+PGtleXdvcmQ+TGVjdGlucy8qaW1t

dW5vbG9neTwva2V5d29yZD48a2V5d29yZD5MZXVrZW1pYSwgQi1DZWxsPC9rZXl3b3JkPjxrZXl3

b3JkPkx5bXBob21hLCBCLUNlbGw8L2tleXdvcmQ+PGtleXdvcmQ+TWljZTwva2V5d29yZD48a2V5

d29yZD5NaWNlLCBOdWRlPC9rZXl3b3JkPjxrZXl3b3JkPk1pY2UsIFNDSUQ8L2tleXdvcmQ+PGtl

eXdvcmQ+TW9sZWN1bGFyIENvbmZvcm1hdGlvbjwva2V5d29yZD48a2V5d29yZD5TaWFsaWMgQWNp

ZCBCaW5kaW5nIElnLWxpa2UgTGVjdGluIDI8L2tleXdvcmQ+PGtleXdvcmQ+U3Vydml2YWwgQW5h

bHlzaXM8L2tleXdvcmQ+PGtleXdvcmQ+VHJhbnNwbGFudGF0aW9uLCBIZXRlcm9sb2dvdXM8L2tl

eXdvcmQ+PGtleXdvcmQ+VHVtb3IgQ2VsbHMsIEN1bHR1cmVkPC9rZXl3b3JkPjwva2V5d29yZHM+

PGRhdGVzPjx5ZWFyPjIwMDU8L3llYXI+PHB1Yi1kYXRlcz48ZGF0ZT5NYXkgMTU8L2RhdGU+PC9w

dWItZGF0ZXM+PC9kYXRlcz48aXNibj4xMDc4LTA0MzIgKFByaW50KSYjeEQ7MTA3OC0wNDMyIChM

aW5raW5nKTwvaXNibj48YWNjZXNzaW9uLW51bT4xNTg5NzU4OTwvYWNjZXNzaW9uLW51bT48dXJs

cz48cmVsYXRlZC11cmxzPjx1cmw+aHR0cDovL3d3dy5uY2JpLm5sbS5uaWguZ292L3B1Ym1lZC8x

NTg5NzU4OTwvdXJsPjwvcmVsYXRlZC11cmxzPjwvdXJscz48ZWxlY3Ryb25pYy1yZXNvdXJjZS1u

dW0+MTAuMTE1OC8xMDc4LTA0MzIuQ0NSLTA0LTIyOTA8L2VsZWN0cm9uaWMtcmVzb3VyY2UtbnVt

PjwvcmVjb3JkPjwvQ2l0ZT48L0VuZE5vdGU+

ADDIN EN.CITE.DATA [9]. SUPPLEMENTARY REFERENCES ADDIN EN.REFLIST Charbonneau B, Vogel RI, Manivel JC, Rizzardi A, Schmechel SC, Ognjanovic S, et al. Expression of FGFR3 and FGFR4 and clinical risk factors associated with progression-free survival in synovial sarcoma. Hum Pathol. 2013;44:1918-26.Rizzardi AE, Johnson AT, Vogel RI, Pambuccian SE, Henriksen J, Skubitz AP, et al. Quantitative comparison of immunohistochemical staining measured by digital image analysis versus pathologist visual scoring. Diagnostic pathology. 2012;7:42.Koopmeiners JS, Modiano J. A Bayesian adaptive Phase I-II clinical trial for evaluating efficacy and toxicity with delayed outcomes. Clin Trials. 2014;11(1):38-48.Thall, PF, Cook, JD. Dose-Finding Based on Efficacy-Toxicity Trade-Offs. Biometrics. 2004;60 (3): 684-693.Guidance for Industry. Good Clinical Practice (VICH GL9). Washington, DC: US Dept of Health and Human services FDA, Center of Vet Med; 2001.Oken MM, Creech RH, Tormey DC, Horton J, Davis TE, McFadden ET, Carbone PP. Toxicity and Response Criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol. 1982;5:649-655. Schappa JT, Frantz AM, Gorden BH, Dickerson EB, Vallera DA, Modiano JF. Hemangiosarcoma and its cancer stem cell subpopulation are effectively killed by a toxin targeted through epidermal growth factor and urokinase receptors. Int J Cancer 2013;133(8):1936-44.Kreitman RJ,?Tallman MS,?Robak T,?Coutre S,?Wilson WH,?Stetler-Stevenson M,?et al. Phase I trial of anti-CD22 recombinant immunotoxin moxetumomab pasudotox (CAT-8015 or HA22) in patients with hairy cell leukemia. J Clin Oncol. 2012;30(15):1822-8.Vallera DA, Todhunter DA, Kuroki DW, Shu Y, Sicheneder A, Chen H. A bispecific recombinant immunotoxin, DT2219, targeting human CD19 and CD22 receptors in a mouse xenograft model of B-cell leukemia/lymphoma. Clin Cancer Res. 2005;11:3879-88.Supplementary Table 1. Correlation between patient covariates and deathVariableNHR (95% CI)p-valueAge (N = 212) > 61 years 104 1.66 (1, 2.74) 0.048≤ 61 years 108 Gender (N = 212) Male 95 1.07 (0.65, 1.76) 0.802Female 117 Race = (N = 204) White 183 0.91 (0.33, 2.5) 0.848Non-White 21 Tumor Volume (N = 146) > 550 mm3 73 2.77 (1.33, 5.79) 0.007≤ 550 mm3 73 Metastasis (N = 134) Yes 41 2.31 (1.23, 4.35) 0.009No 93 Tumor Site (N = 212) Upper abdomen or retroperitoneum 81 1.42 (0.86, 2.33) 0.17Any other location 131 EGFR (N = 212) > 410 FPKMs 106 1.69 (1.02, 2.81) 0.043≤ 410 FPKMs 106 PLAUR (N = 212) > 757 FPKMs 106 1.63 (0.98, 2.69) 0.058≤ 757 FPKMs 106 *The TCGA included samples from 59 patients with dedifferentiated liposarcomas, 2 patients with desmoid tumors, 1 patient with an undifferentiated pleomorphic sarcoma with giant cells, 105 patients with leiomyosarcomas (LMS), 9 patients with malignant peripheral nerve sheath tumors (MPNST), 25 patients with myxofibrosarcomas, 50 patients with undifferentiated pleomorphic sarcomas, and 10 patients with synovial sarcomas.Supplementary Table 2: Patient information (TMA)CovariateUnits/LevelMean (SD) or N(%)Age Years36.9 (17.1)Sex Male27 (61.4) Female17 (38.6)Subtype Monophasic29 (65.9) Biphasic15 (34.1)Tumor size 5 cm or less14 (31.8) Greater than 5 cm29 (65.9) Unknown1 (2.3)Lymph Node Involvement No43 (97.7) Yes1 (2.3)Metastasis at Diagnosis No41 (93.2) Yes3 (6.8)Nodes or Metastasis No40 (90.9) Yes4 (9.1)Pre-Surgery Treatment None32 (72.7) Chemotherapy7 (15.9) Radiation4 (9.1) Chemotherapy and Radiation1 (2.3)Chemotherapy No21 (47.7) Yes23 (52.3)Radiation No13 (29.5) Yes30 (68.2) Unknown1 (2.3)Supplementary Table 3: Cox regression analysis for time-to-progressionCovariateUnivariate ModelMultivariate Model with markers onlyMultivariate Model with markers plus other covariatesHazard Ratiop-valueHazard Ratiop-valueHazard Ratiop-valueEGFR expression 0.92 (0.76, 1.11)0.3690.86 (0.68, 1.1)0.2410.9 (0.71, 1.14)0.401uPAR expression 1.02 (0.8, 1.29)0.9021.13 (0.85, 1.5)0.3970.95 (0.68, 1.33)0.775Age 1 (0.96, 1.03)0.903Sex (reference = Male) 0.64 (0.19, 2.14)0.464Subtype (reference = monophasic) 1.12 (0.36, 3.51)0.848Tumor Size (reference = 5 cm or less) 3.2 (0.84, 12.25)0.089Nodes or Metastasis (reference = no) 4.04 (0.41, 40.26)0.234Chemotherapy (reference = no) 0.49 (0.11, 2.11)0.336Radiation (reference = no) 0.7 (0.22, 2.29)0.558Supplementary Figure 1. Survival probability based on EGFR expression 47814621871330P=0.0434000020000P=0.043 Supplementary Figure 1. Survival probability based on EGFR expression. Graph illustrating death events in patients where EGFR expression was in the upper 50th percentile versus patients with EGFR expression in the lower 50th percentile.Supplementary Figure 2. Quantification of EGFR and uPAR expression in normal canine tissues and in canine hemangiosarcomaABSupplementary Figure 2. Quantification of EGFR and uPAR expression in normal canine tissues and in canine hemangiosarcoma. Box and whisker plots summarizing EGFR (A) and uPAR (B) expression in a tissue microarray containing kidney, liver, and spleen tissues from normal dogs, tumor samples from 15 dogs in the SRCBST-1 study, and samples from two dogs with focal splenic hematomas. Supplementary Figure 3. CONSORT diagram for SRCBST study1063385415Total enrolled (n=23)Euthanized prior to doxorubicin due to metastasis and hemoabdomen (n=1)Safety and toxicity assessment (n=23)Follow-up >180 days until death (n=14)???ExcludedBenign splenic lesions or non HSA tumors, excluded (n=31)Splenic HSA (n=20)Metastasis at surgery, excluded (n=10)Screened (n=10)Declined screening, excluded (n=4)Screened (n=6)Metastasis, excluded (n=1)Splenic HSA (n=28)Screened (n=28)Concurrent cardiac disease, excluded (n=1)Declined study participation, excluded (n=2)Metastasis, excluded (n=7) Enrolled (n=5)Enrolled (n=18)Splenectomy at the VMC(n=51)Splenectomy at rDVM, referral to the VMC(n=28)Initial Contacts (n=181)Excluded from initial contact (n=102)Did not meet inclusion criteria (n=50)Declined participation (n=52)Alive <180 days (n=9)0Total enrolled (n=23)Euthanized prior to doxorubicin due to metastasis and hemoabdomen (n=1)Safety and toxicity assessment (n=23)Follow-up >180 days until death (n=14)???ExcludedBenign splenic lesions or non HSA tumors, excluded (n=31)Splenic HSA (n=20)Metastasis at surgery, excluded (n=10)Screened (n=10)Declined screening, excluded (n=4)Screened (n=6)Metastasis, excluded (n=1)Splenic HSA (n=28)Screened (n=28)Concurrent cardiac disease, excluded (n=1)Declined study participation, excluded (n=2)Metastasis, excluded (n=7) Enrolled (n=5)Enrolled (n=18)Splenectomy at the VMC(n=51)Splenectomy at rDVM, referral to the VMC(n=28)Initial Contacts (n=181)Excluded from initial contact (n=102)Did not meet inclusion criteria (n=50)Declined participation (n=52)Alive <180 days (n=9)Supplementary Figure 3. Enrollments, exclusions, and assessments. Flow chart with details of dogs enrolled in the study and exclusions from each of the measured endpoints. ................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download