Download Supplementary Appendix Table of Contents: Supplementary

Supplementary Appendix
Table of Contents:
Supplementary methods……………………………………………………….2-4
Supplementary results………………………………………………………….4
Figure 1: Mean renal function…………………………………………………5
Table 1: Acute rejection specifics……………………………………………..6-7
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Methods:
Inclusion and Exclusion Criteria
Patients were also excluded if they were recipients of an ABO incompatible donor kidney, a multiple organ
transplant, or if the donor or recipient were known to be seropositive for human immunodeficiency virus (HIV),
hepatitis C or B; except for hepatitis B surface antibody positivity. Patients with thrombocytopenia (<75,000/mm 3),
an absolute neutrophil count (ANC) of < 1,000/mm3); leucopoenia (< 2,000/mm3), and/or anemia (hemoglobin < 6
g/dL) prior to study enrollment were also excluded. Patients could not be taking or have been taking an
investigational drug within the 30 days prior to transplant or have a known hypersensitivity to tacrolimus,
mycophenolate mofetil, RATG, daclizumab, basiliximab or corticosteroids. Those patients with severe diarrhea or
any other gastrointestinal disorders that might interfere with their ability to absorb oral medications were also
ineligible for inclusion. Patients must be free from malignancy for at least five years, except for successfully excised
squamous or basal cell carcinoma of the skin. Female recipients of childbearing potential must have a negative
pregnancy testing within the past 48 hours of study inclusion and be willing to use two forms of contraception
simultaneously from the beginning of study enrollment through the duration of the study and for six weeks
following the study. PRA screening was done using FlowPRA® . Single Antigen (SA) beads analysis was
performed using the LABScreen® assay and Luminex platform (One Lambda). Signal amplification was performed
using biotinylated goat anti-human IgG secondary antibodies and StreptAvidin-PE. Analysis of the HLA antibodies
pattern and epitopes sequence analysis was performed using ClustalW2 multiple sequence alignment program for
proteins (EMBL-EBI). The presence or absences of shared epitopes are subsequently confirmed by performing Tand B-cells flow cytometry cross-smatch using surrogate blood donors. Although the signal strength is considered a
major determinant regarding the clinical relevance of antibodies identified by single antigen beads, the pattern of
reactivity should not be overlooked. Signal strength alone is not a sufficient metric to assess the potential of a socalled weak antibody to result in a positive cross-match. In the analysis of single antigen beads data for patients,
special consideration should be given to the antibodies that may be directed against public, such as Bw4 and Bw6,
shared epitopes. This includes s antibodies directed to HLA B-locus cross-reactive groups (CREGs), or antibodies
to HLA DP locus. The signal may look artificially low since these epitopes are distributed across many beads.
2
Induction therapy:
The initial intra-operative dose of rabbit anti-thymocyte globulin was administered one hour after the initial
methylprednisolone dose. Subsequent doses were administered over a minimum of 4 hours and the day of discharge
dose was delayed until the following clinic day to facilitate discharge. Rabbit anti-thymocyte globulin dosage
adjustments were based on package insert guidelines. These guidelines indicates no dosage adjustment for white
blood cell count (WBC) > 3000 cells/µL or platelets >75,000 cells/µL, 50% dose reduction for a WBC between
3000 and 2000 cells/µL or platelets between 50,000 and 75,000 cells/µL. Doses were held for WBC <2000 cells/µL
and platelets <50,000 cells/µL.
Maintenance Immunosuppression and Antimicrobial Prophylaxis
Tacrolimus trough concentration were drawn and evaluated on at a minimum of post-operative days 3, 5 and 7,
weeks 2, 3, 4 and months 2, 3, 6, 9, and 12 or more frequently as clinically indicated. Tacrolimus toxicity was
treated by reducing the tacrolimus exposure to the lower end of the desired target range, unless the toxicity persisted;
then lower exposure was approved by the primary investigator. Mycophenolate mofetil dose adjustments were
strongly discouraged within the first month post-transplant and were only allowed based on adverse drug events or
toxicity at the discretion of the primary investigator. Mycophenolate mofetil dosage adjustments were based on the
following algorithm: dose adjustments for leucopenia were allowed; if the WBC was between 2000 and 3000
cells/µL the patient’s current dose could be reduced by 25%, if the WBC was <2000 with ANC >1000 cells/µL dose
could be reduced by 50%. Doses could be held if the ANC<1000 cells/µL. Corticosteroids were initiated in the
operating room with 500 mg of methylprednisolone administered intravenously, followed by 250 mg on day 1, 125
mg on day 2, prednisone 50 mg on day 3, prednisone 20 mg daily on days 4 thru 30, prednisone 10 mg day 30 to 45,
and prednisone 5 mg thereafter.
Prophylaxis against opportunistic infection included the following regimen: prophylactic antifungal therapy
consisted of nystatin oral suspension for 30 days post-transplant, sulfamethoxazole/trimethoprim daily for 90 days
and valganciclovir for cytomegalovirus (CMV) prevention. Recipients who were seropositive for CMV at the time
of transplant received valganciclovir for 90 days adjusted for renal function based on package insert guidelines.
Recipients who were seronegative for CMV at the time of transplant but received a seropositive kidney received
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valganciclovir for 180 days. Those patients not at risk for CMV received acyclovir for prevention of other herpes
viruses.
Efficacy and Safety End Points
BK viral PCRs were obtained at a minimum at 1, 2, 3, 6, 9, and 12 months or more frequently if viremia was
detected. Leucopenia was defined as a total white blood cell count of less than 2,000 cells/mm3 and neutropenia,
defined as an absolute neutrophil count of less than 1,000 cells/mm3 and need for colony stimulating factors and
incidence of thrombocytopenia, defined as a platelet count of less than 100,000 cells/mm 3.
Results
The incidence of leucopenia within the first 7 days post-transplant was low and occurred in 5 patients in the IL2RA
group versus 8 in the RATG group. The incidence of leucopenia after 7 days post-transplant was 2% in the IL2RA
group versus 4% in the rabbit anti-thymocyte group (p=0.58). However, thrombocytopenia occurred in 14% of the
IL2RA group within the first 7 days versus 37% in the RATG (p<0.003). After 7 days the incidence of
thrombocytopenia fell to 4% in the IL2RA group and 8% in the RATG (p=0.37).
4
5
Table 1: Acute Rejection Specifics
Induction PRA
IL2RA
37
IL2RA
0
IL2RA
80
IL2RA
0
IL2RA
46
IL2RA
0
Race
Black
Black
Black
Black
Black
CIT>24
hours
1
0
1
0
1
NonBlack
0
Banff
Time to
NonGrade rejection (days) compliance
1B
87
No
1B
277
Yes
2A
4
No
2A
10
No
2A
8
No
1A
80
No
Medication
PAK
Intolerance
No
No
No
No
No
No
No
No
No
No
Yes-MMF held
during admission
for afib, bacteremic
with persistent
headaches r/o
No
malignancy
Non-
Average FK
Level
(ng/mL)*
9.6±8.7
5.7±5.2
6±3.4
5.8±3.2
5.6±3.8
Day 365 Scr (mg/dL)
2.0
2.2
1.2
1.4
1.5
11.6±3.4
1.4
9±3.2
0.9
10.9±1.2
2.0
6±5.3
1.3
7.2±2.2
1.7
9.8±4.7
1.5
Yes-MMF
IL2RA
0
Black
0
1A
329.00
No
Yes
IL2RA
21
Black
0
1B
97
No
No
IL2RA
0
Black
0
1A
22
Yes
No
IL2RA
0
NonBlack
0
III
62
No
No
RATG
0
NonBlack
0
2A
154
No
No
RATG
0
NonBlack
0
2A
287
Yes
No
intolerance GI
Converted to
everolimus
following CNI
toxicity on Bx, also
at same visit MMF
reduced for
diarrhea, EVR levels
not above 3 prior to
rejection
Biopsy 7 days prior
no rejection,
patient was taking
incorrect dose of
prednisone 10 mg
vs 20 mg
Protocol bx
revealed Borderline
1 month before
treated with SM x
3, also was
converted to rapa
for tremor between
4 and 6 weeks rapa level at 6
weeks was 4 then
12.1 at time of
rejection, patient
later converted
back to FK for rapa
induced mouth
ulcers
MPA reduced
secondary to BK
prior to rejection
event, also ACS
with cardiac cath
Was taking MMF
250 daily by
mistake, but also
MMF held prior to
rejection secondary
to groin mass which was found to
be one of many dxn metastatic
squamous cell CA
8.5±0.1
6
RATG
0
Black
0
1A
341
No
No
Conversion back to
everolimus from
tacrolimus
following hip
surgery
2.7**
2.9
Recurrent UTIs,
NonRATG
0
Black
EVR+ MMF 500 mg
0
1A
219
No
No
BID,
7.8±0.75
3.5
Diarrhea 4 months
before - MMF held,
then restarted at
reduced dose. Also
BK PCR positive
prior - conversion
RATG
37
Black
1
1A
178
No
No
to leflunomide
4.7±5.9
1.6
Lost third party
coverage and did
not feel like coming
to his visits
anymore - was out
of medication for
approximately 1
Nonweek per report
RATG
33
Black
1
1B
266
Yes
No
prior to biopsy
None
Graft loss
*for rejections>90 days - three values prior to rejection, noncompliance defined by evidence documenting noncompliance in clinic notes **everolimus
level
7