carcinoma renal

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2. Immunomodulatory activity of nivolumab in
previously treated and untreated metastatic
renal cell carcinoma (mRCC): Biomarker
correlations with clinical outcomes
Toni K. Choueiri,1 Mayer N. Fishman,2 Bernard Escudier,3 David F. McDermott,4 Harriet Kluger,5
Walter M. Stadler,6 Jose Luis Perez-Gracia,7 Douglas McNeel,8 Brendan Curti,9 Michael R.
Harrison,10 Elizabeth R. Plimack,11 Leonard Appleman,12 Lawrence Fong,13 Charles G. Drake,4
Tina C. Young,14 Scott D. Chasalow,14 Petra Ross-Macdonald,14 Jason S. Simon,14
Dana Walker,14 Mario Sznol5
Abstract 4500
1Dana-Farber Cancer Institute, Boston, MA, USA; 2H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, USA; 3Institut
Gustave Roussy, Villejuif, France; 4Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, MD,
USA; 5Yale Cancer Center, New Haven, CT, USA; 6University of Chicago Medicine, Chicago, IL, USA; 7University Clinic of
Navarra, Pamplona, Spain; 8University of Wisconsin-Madison, Department of Medicine, Madison, WI, USA; 9Providence Cancer
Center, Providence Portland Medical Center, Portland, OR, USA; 10Duke University Medical Center, Durham, NC, USA; 11Fox
Chase Cancer Center, Philadelphia, PA, USA; 12University of Pittsburgh Medical Center (UPMC) Cancer Pavilion, Pittsburgh,
PA, USA; 13University of California San Francisco (UCSF) Helen Diller Family Comprehensive Cancer Center, San Francisco,
CA, USA; 14Bristol-Myers Squibb, Princeton, NJ, USA

3. Introduction
• Nivolumab, a fully human anti-PD-1 immune checkpoint inhibitor, provided a
median overall survival (OS) of 18.2–25.5 months in patients with previously
treated mRCC1–2
– Median OS ≤16.5 months has been reported for currently available therapies in
previously treated mRCC2
• Nivolumab has demonstrated immunomodulatory effects consistent with
PD-1 inhibition3
– Tumor-associated lymphocytes increased in on-treatment biopsies
– Concentrations of IFNγ–stimulated cytokines increased in serum
• Analyses in this exploratory study will provide insight into potential
predictive biomarkers for nivolumab in mRCC
3
PD, programmed death; IFNγ, interferon gamma; mRCC, metastatic renal cell carcinoma.
1. Harshman LC, et al. Cancer Immunol Res. 2014;2:1132–41. 2. Motzer RJ, et al. J Clin Oncol. 2015;33:1430–7. 3.
Choueiri T, et al. J Clin Oncol. 2014;32(15 suppl). Abstract 5012.

4. mRCC (clear cell) after anti-
angiogenic therapy
(n = 67)
• 1–3 prior therapies
• Progressed from most recent
therapy within 6 months
• KPS ≥70%
Treatment-naïve mRCC (clear cell)
(n = 24)
• KPS ≥70%
R
1:1:1 Treat until CR,
progression or
intolerable toxicity
Arm 1
Nivolumab 0.3 mg/kg IV Q3W
Arm 2
Nivolumab 2.0 mg/kg IV Q3W
Arm 3
Nivolumab 10.0 mg/kg IV Q3W
Arm 4
Nivolumab 10.0 mg/kg IV Q3W
Study design and objectives
• Primary objective: To assess the pharmacodynamic activity of nivolumab on tumor-infiltrating T cells
and serum chemokines in patients with metastatic clear-cell RCC
• Key secondary objectives: To assess antitumor activity, safety, and tolerability
• Key exploratory objectives: To assess associations between PD-L1 expression, serum cytokines,
gene expression, TCR repertoire, and efficacy
4
4
Clinical Trials Registry: NCT01358721.
CR, complete response; KPS, Karnofsky performance status; Q3W, every 3 weeks; R, Randomization; TCR, T-cell receptor.

5. Analysis Sample Assay Time points
PD-L1 expression
FFPE tumor
biopsy
Immunohistochemistry
(BMS/Dako; 28-8 antibody) Baseline
Soluble factors Serum Luminex™ (Myriad RBM) Baseline, C2D8
Gene expression profiling Tumor biopsy Affymetrix® Human Genome U219 Baseline, C2D8
T-cell receptor sequencing
Frozen tumor
biopsy, PBMC
immunoSEQ™ (Adaptive
Biotechnologies)
Baseline, C2D8
5
Assessments of
immunomodulatory activity
Baseline refers to tumor biopsies collected during screening or peripheral blood collected on day 1 of the first cycle, prior to nivolumab
infusion.
C2D8, cycle 2 day 8; FFPE, formalin-fixed, paraffin-embedded; PBMC, peripheral blood mononuclear cells; PD-L, programmed death ligand.

6. Nivolumab mechanism of action
6
Expected associations with better outcome
• ↑ Serum soluble markers of IFNγ signaling
• ↑ IFNγ–regulated gene expression
• ↑ T-cell clonality in tumor
MHC
PD-L1
PD-1
PD-1
T-cell
receptor
PD-L2
T cell
NFκB
Other
PI3K
Tumor cell
IFNγ
IFNγR
Shp-2
Nivolumab
IFNγR, interferon gamma receptor; MHC, major histocompatibility complex.

7. Patient demographics and
baseline characteristics
Previously treated
All (n = 67)
Treatment-naïve
(n = 24)
Total
(N = 91)
Median age, years 61.0 63.5 61.0
Sex, n (%)
Male 46 (69) 15 (63) 61 (67)
Female 21 (31) 9 (38) 30 (33)
Prior therapy, n (%)
Surgery 64 (96) 23 (96) 87 (96)
Radiotherapy 25 (37) 5 (21) 30 (33)
Prior systemic therapy for RCC 67 (100) 0 67 (74)
7

8. aPoint estimates are derived from Kaplan-Meier analyses; 95% CIs are derived from Greenwood’s formula.
CI, confidence interval; NR, not reached.8
Overall survival
0 3 6 9 12 15 18 21 24 27 30 33 36
Overall Survival (Months)
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
ProbabilityAlive
Treatment
group
Events/
Patients
Median OS
(95% CI)a
0.3 mg/kg 12/22 16.4 (10.1–NR)
2 mg/kg 9/22 NR
10 mg/kg 11/23 25.2 (12.0–NR)
10 mg/kg
(naïve)
7/24 NR
12 months
75 (64–83)
24 months
58 (46–68)
OS rate, % (95% CI)a:
Total

9. Overall survival by PD-L1 expression
9
12 months
71 (44–87)
71 (52–83)
24 months
64 (37–82)
48 (30–64)
OS rate, % (95% CI)a:
PD-L1 expression ≥5%
PD-L1 expression <5%
0 3 6 9 12 15 18 21 24 27 30 33 36
Overall Survival (Months)
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
ProbabilityAlive
aPD-L1 expression by tumor membrane staining; bPoint estimates are derived from Kaplan-Meier analyses;
95% CIs are derived from Greenwood’s formula.
PD-L1
expression
Events/
Patients
Median OS,
(95% CI)b
≥5% 6/18 NR
<5% 21/38 23.4 (13.1–33.3)

10. Serum soluble factors and overall survival
Cox proportional hazards model accounts for dose and naïve/previously treated status.
Sample sizes range from 74‒90, and HRs are for comparing Q3 vs Q1.10
Baseline Change from Baseline
ICAM1
VCAM1
VEGF
CXCL9
ICAM1
VCAM1
TIMP1
VEGF
CXCL9
Recognized prognostic markers
CXCL9
TIMP1
log (HR)
Higher = longer OS Lower = longer OS
log (HR)
Higher = longer OS Lower = longer OS
1.1 (0.8, 1.6)
1.2 (1.1, 1.5)
1.5 (1.1, 2.0)
1.4 (1.1, 1.9)
1.3 (1.0, 1.6)
0.7 (0.5, 1.1)
1.0 (0.9, 1.3)
1.2 (0.7, 2.0)
1.2 (0.9, 1.6)
1.0 (0.6, 1.7)

11. Gene expression and tumor burden reduction:
Baseline
311Genesa
n = 13n = 43
Lower expression
•Establishment of protein localization (P < 10–5)
•Negative regulation of epithelial cell proliferation involved in
lung morphogenesis (P < 10–4)
•Genes downregulated by ipilimumab in melanoma1 (P < 10–4)
Higher expression
•Genes upregulated by ipilimumab in melanoma1 (P < 10–23)
•Immune system (45 genes; P < 10–7): eg, IL15RA, IL1R2, IRF1
•Myeloid lineage: eg, IL1A, LINC00158, PRAM1, SPI1
•Lymphoid lineage: eg, CD3E, AIM2, GZMB, NKG7, CD7,
CTSW
Z score
-2SD +2SD
60
40
20
0
-20
-40
-60
-80
-100
≥20% reduction in tumor burden
11
Underlining denotes Gene Ontology Biological Process category.
1. Ji RR, et al. Cancer Immunol Immunother. 2012;61:1019‒31.
aP < 0.01, >1.3-fold, false discovery rate <16%.
BestΔtumor
burden(%)

12. Gene expression and tumor burden reduction:
On treatment
779Genesa
Lower expression
•Cellular component organization (P < 10–18)
•Signaling (P < 10-16)
•Genes downregulated by ipilimumab in melanoma1 (P < 10–5)
Higher expression
•Genes upregulated by ipilimumab in melanoma1 (P < 10–135)
•Immune system (188; P < 10–82)
•Known myeloid lineage (51): eg, CD68, CD86, CASP1, CSF3R
•Known lymphoid lineage (>65): eg, TCRα/β,CD3D,
CD8A, CD28
•Cytolytic function: eg, KLRG1, granzymes, PRF1
•Interferon regulated (24): eg, AIM2, CASP1, CCL8, IRF9
•Immune checkpoint molecules: eg, TIGIT, CTLA-4, PD-L2,
IL10RA
n = 11n = 44
80
60
40
20
0
-20
-40
-60
-80
-100
12
Underlining denotes Gene Ontology Biological Process category.
1. Ji RR, et al. Cancer Immunol Immunother. 2012;61:1019‒31.
Z score
-2SD +2SD
BestΔtumor
burden(%)
aP < 0.01, >1.3-fold, false discovery rate <13%.
ND
≥20% reduction in tumor burden

13. Immune checkpoint gene expression and tumor
burden reduction: On treatment
CTLA, cytotoxic T-lymphocyte antigen; TIGIT, T-cell immunoreceptor with Ig and ITIM domains.
CTLA4 TIGIT
7
6
5
4
3
2
PD-L2
CTLA-4 TIGIT
P = 0.003
RMASignal
P = 0.002
PD-L2
P = 0.002
4
16
64
256
Mean, 95% CI
≥20% reduction
<20% reduction
13

14. T-cell receptor sequencing
14
High clonality TCR repertoire Low clonality TCR repertoire
TCR, T-cell receptor.
• Targeted next-generation sequencing of TCRs amplified
from tumor and blood can provide insight
into the status of the immune system:
– TCR clonality
– % of T cells in the tumor
– Comparisons of the TCR clones present in the blood and tumor
tumor cell
T cell clones

15. T-cell receptor repertoire and overall survival
15 aTCR metrics associated with survival in a penalized multivariable Cox PH model. HR, hazard ratio.
aPre-treatment
On treatment
Pre-treatment
aPre-treatment
On treatment
On treatment
Tumor T-Cell Frequency
Tumor Clonality
Blood Clonality
Univariate Cox proportional hazards analysis
HR (95% CI)
-1.5 -1.0 0.5 0.0 0.5 1.0 1.5
0.5 (0.2, 0.9)
0.7 (0.3, 1.3)
0.7 (0.3, 1.2)
0.8 (0.5, 1.4)
1.2 (0.8, 1.7)
1.1 (0.8, 1.6)
log (HR)
Higher = longer OS Lower = longer OS

16. Summary and conclusions
• In this exploratory study, nivolumab provided meaningful survival benefit in patients
with mRCC, consistent with other reports,1 and irrespective of PD-L1 expression
• The following changes in biomarkers, suggestive of adaptive immune activity in
patients with better clinical outcomes, were observed:
• ↑ Serum markers of IFNγ activation
• ↑ Tumor gene expression indicating lymphoid and myeloid infiltration with
active checkpoint regulation
• ↓ Clonality of TCR in blood
• ↑ T-cell counts in tumor
• Significant upregulation of CTLA-4 and PD-L2 while on nivolumab underscores the
potential of nivolumab + ipilimumab combination therapy in mRCC
• Additional investigation in randomized, controlled trials is needed to better
understand the potential for predicting outcomes based on these observed changes
16 1. Motzer RJ, et al. J Clin Oncol. 2015;33:1430–7.

17. Acknowledgments
• The patients and their families
• The study sites enrolling patients to the trial
• Nursing staff and study coordinators
• Support for this work from Bristol-Myers Squibb and Ono Pharmaceutical Co.,
Ltd
• Dako for development of the PD-L1 immunohistochemistry assay
• Ryan Emerson, PhD, Harlan Robins, PhD, Bryan Howie, PhD, from Adaptive
Biotech for the TCR repertoire analysis
• All authors contributed to and approved the presentation; writing and editorial
assistance was provided by S. Thomas, PhD, of PPSI, funded by Bristol-Myers
Squibb
17