SUPPLEMENTAL METHODS

Procedures

Throughout the study, treatment could be temporarily suspended for ≤28 days if the patient

experienced toxicity considered related to each or both treatments. If patients missed >28

consecutive days of scheduled treatment due to adverse events associated with that treatment,

treatment was discontinued. Patients who discontinued one of the study treatments could

continue and receive single-agent treatment at the investigator’s discretion.

To define the patient population with PTEN-loss tumors in the primary and secondary

analyses, PTEN status was assessed by an immunohistochemistry assay performed on archival

tumor tissue at The Institute of Cancer Research, where this assay has been extensively

verified. The assay was a pre-specified analysis and was conducted prior to unblinding. PTEN

protein expression was determined on formalin-fixed and paraffin-embedded sections as

previously described.(1-3) In cases where the prostate cancer specimen was considered

multifocal, PTEN status classification was based on the tumor area with the lowest tumor PTEN

staining. In addition to the primary analysis of PTEN status using The Institute of Cancer

Research platform, PTEN expression by immunohistochemistry (using the SP218 antibody) has

also been determined using an assay developed at Ventana. Molecular profiling of the DNA

extracted from tumor samples was performed after the unblinding at Foundation Medicine Inc.,

using the FoundationOne next-generation sequencing–based platform.(4) The cutoff used for

PTEN loss was homozygous and heterozygous loss of PTEN and single-nucleotide variants

predicted to be deleterious in combination with loss of heterozygosity. Genetic loss of PTEN by

dual-color fluorescence in situ hybridization (FISH) was performed at a central laboratory (Core

Diagnostics, Palo Alto, CA). The PTEN DNA probe is a 180 Kb SpectrumOrange directly

labeled, fluorescent DNA probe specific for the PTEN gene locus (10q23). The Chromosome

Enumeration Probe (CEP) 10 DNA probe is a SpectrumGreen directly labeled, fluorescent DNA

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probe specific for the alpha satellite DNA sequence at the centromeric region of chromosome

10 (10p11.1-q11.1). Hybridization of the PTEN FISH probes was viewed using a fluorescence

microscope equipped with appropriate excitation and emission filters allowing visualization of

the intense orange and green fluorescent signals. Enumeration of the PTEN and CEP 10

signals was conducted by microscopic examination of the nucleus. A minimum of 100 tumor

cells were examined on each specimen, and the average number of PTEN and CEP signals/cell

was determined for each specimen.

Statistical analysis

The 90% CIs for the hazard ratio were calculated with the expectation that for clinically

meaningful outcomes, the hazard ratio would be lower than 1, and the upper of limit of the two-

sided 90% CI would be close to 1.0. Approximately 80 randomized patients per treatment

cohort, or 240 randomized patients overall, were planned for enrollment. Assuming 60%

prevalence of PTEN loss in prostate cancer, there was projected to be 144 total (96 per

comparison) patients with PTEN-loss tumors.

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APPENDIX FIGURES

Figure A1. (A) Summary of the source of prostate specimens provided, (B) distribution of the age of the tumor specimen, (C) PTEN-loss concordance of the diagnostic classifications between the multiple IHC and DNA-based platforms and (D) PTEN IHC score distributions for the ICR and Ventana assays. FISH, fluorescence in situ hybridization; HOM, homozygous deletion; HET, heterozygous deletion; ICR, The Institute of Cancer Research; IHC, immunohistochemistry; NGS, next-generation sequencing; PTEN, phosphatase and tensin homolog; SNV+LOH, deleterious mutation with concomitant loss of heterozygosity.

A

Tissue Submitted Number Patients

Primary 220

Metastatic 18

Duplicate: 1 primary and 1 Metastatic 3

Data not available 12

Total 253

B

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41

42434445464748

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C

D

PTEN IHC Score Distribution – ICR Assay

1 9 17 25 33 41 49 57 65 73 81 89 97 1051131211290%

20%

40%

60%

80%

100%

ICR_3ICR_2ICR_1ICR_0

PTEN IHC Score Distribution – Ventana Assay

1 10 19 28 37 46 55 64 73 82 91 100 109 118 127 1360%

20%

40%

60%

80%

100%

Ven_3Ven_2Ven_1Ven_0

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Figure A2. Study design. ECOG PS, ECOG Scale of Performance Status; PO, by mouth; QD, once daily; R, randomization. a Abiraterone 1000 mg with prednisone/prednisolone 5 mg twice daily. b In the placebo cohort, patients were further randomized in a 1:1:1 ratio to receive ipatasertib 400 mg QD/placebo and 200 mg QD/placebo.

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Figure A3. Subgroup analysis of radiographic progression-free survival for the ipatasertib 400-mg cohort. ALK-P, alkaline phosphatase; ECOG, Eastern Cooperative Oncology Group; HR, hazard ratio; ITT, intent to treat; PSA, prostate-specific antigen.

a Unstratified HR.

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Figure A4. Oncoprint of individual gene alterations clustered by signaling pathway association in the total NGS-evaluable population (N=89; Figures A, C) and the segment of the NGS-evaluable population that was classified as PTEN non-loss by the ICR IHC assay (N=39; Figures B, D). Samples were ranked based on duration of PFS (A, B) or ORR (C, D).

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APPENDIX TABLES

Table A1. Summary of Adverse Events in the Phase 1b Part of the A.MARTIN Study

AE, n (%)Ipatasertib 400 mg +

Abiraterone

(n=14)

Total, any AE 14 (100)

Diarrhea 10 (71.4)

Vomiting 9 (64.3)

Fatigue 9 (64.3)

Hyperglycemia 7 (50.0)

Nausea 6 (42.9)

Decreased appetite 5 (35.7)

Asthenia 4 (28.6)

Dizziness 3 (21.4)

Back pain 2 (14.3)

Arthralgia 2 (14.3)

Urinary tract infection 2 (14.3)

Pain in extremity 2 (14.3)

Bone pain 1 (7.1)

Rash 1 (7.1)

Pyrexia 1 (7.1)

Hot flush 1 (7.1)

Hypertension 1 (7.1)

Constipation 1 (7.1)

Pain 1 (7.1)

Musculoskeletal pain 1 (7.1)

Dyspnea 0

Insomnia 0

AE, adverse event.

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Table A2. Components of Radiographic Progression(5)

Response Progression

Soft tissue mass Per RECIST 1.1 Per RECIST 1.1

Bone

NA <12 weeks after randomization ≥2 new bone lesions

plus 2 additional at confirmation (“2+2”) on a second bone scan ≥4 weeks later

≥12 weeks after randomization ≥2 new bone lesions

consistent with progression (without need for confirmatory bone scan)

The date of progression is the date of the first scan that shows the change.

NA, not applicable; RECIST, Response Evaluation Criteria In Solid Tumors.

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Table A3. PTEN Assays

PTEN Assay Sample SizeAntibody

Assay Laboratory

Cutoff for PTEN Loss

PTEN Loss Prevalence

Protein based

ICR IHC (1, 2) 165

CST 138G6

ICR

IHC 0 = 100% of

tumor43%

Ventana IHC 146

SP218

Ventana Medical Systems

IHC 0 ≥50% of tumor 56%

DNA basedFISHa 196 Core

Diagnostics

HO, HE loss in ≥30% of

tumor36%

NGS 89FoundationOne

FMI

HO, HE loss

SNV+LOH44%

FISH, fluorescence in situ hybridization; FMI, Foundation Medicine Inc; HE, heterozygous loss; HO, homozygous loss; ICR, The Institute of Cancer Research; IHC, immunohistochemistry; LOH, loss of

heterozygosity; NGS, next-generation sequencing; PTEN, phosphatase and tensin homolog; SNV, single-nucleotide variants predicted to be deleterious. All samples were evaluated in a blinded manner.a A minimum of 100 tumor cells were scored in each specimen.

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Table A4. PSA Response Rate

Ipatasertib 400 mg + Abiraterone

(n=84)

Ipatasertib 200 mg + Abiraterone

(n=86)

Placebo +

Abiraterone

(n=83)No. of evaluable patients 84 86 83

Responders, n (%) 90% CI

31 (36.9)28.11 to 46.40

29 (33.7)25.29 to 42.93

29 (34.9)26.25 to 43.75

Difference in responserate vs placebo, %90% CI

1.97−10.25 to 14.18

−1.22−13.24 to 10.80 –

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Table A5. Objective Response Rate by Response Evaluation Criteria In Solid Tumors and

PTEN-loss and PTEN-non loss tumor populations

Ipatasertib 400 mg + Abiraterone

(n=84)

Ipatasertib 200 mg + Abiraterone

(n=86)

Placebo +

Abiraterone

(n=83)No. of evaluable patients 37 39 35

Responders, n (%) 90% CI

12 (32.4)20.56 to 46.41

9 (23.1%)13.69 to 35.63

8 (22.9%)11.91 to 36.46

Complete response, n (%) 2 (5) 1 (3) 2 (6)

Partial response, n (%) 10 (27.0) 8 (20.5) 6 (17.1)

Difference in responserate vs placebo, % 90% CI

9.58

−7.65 to 26.80

0.22

−15.89 to 16.33–

Primary AnalysisICR IHC

PTEN loss PTEN non-lossIpatasertib 400 mg + AbirateronePatients, n 9 15Responders, n (%)90% CI

1 (11.1)1.16, 39.09

4 (26.7)12.18, 50.00

Difference in responserate vs placebo, %90% CI

-3.17-30.93, 24.58

1.67-22.96, 26.29

Ipatasertib 200 mg + AbirateronePatients, n 15 12Responders, n (%)90% CI 4 (26.7) 2 (16.7)

4.52, 39.84Difference in responserate vs placebo, %90% CI

12.38-16.36, 41.12

-8.33-32.14, 15.47

Placebo + AbirateronePatients, n 7 20Responders, n (%)90% CI

1 (14.3)1.49, 50.00

5 (25.0)12.58, 44.20

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Table A6. CTC Conversion in ITT and PTEN-loss and PTEN-non loss tumor populations

Ipatasertib 400 mg + Abiraterone

(n=84)

Ipatasertib 200 mg + Abiraterone

(n=86)

Placebo +

Abiraterone

(n = 83)No. of evaluable patients 41a 47a 48a

Responders, n (%)90% CIb

18 (43.9)31.18 to 57.87

22 (46.8)34.48 to 59.72

20 (41.7)29.59 to 54.55

Difference in responserate vs placebo, % 90% CI

2.24

–15.07 to 19.54

5.14

–11.60 to 21.88–

Primary AnalysisICR IHC

PTEN loss PTEN non-lossIpatasertib 400 mg + AbirateronePatients, n 14 12Responders, n (%)90% CIb 5 (35.7) 8 (66.7)

(39.84, 84.58)Difference in responserate vs placebo, %90% CI

-22.62-54.11, 8.87

34.857.14, 62.56

Ipatasertib 200 mg + AbirateronePatients, n 15 18Responders, n (%)90% CIb

11 (73.3)50.00, 87.82

4 (22.2)1.06, 41.88

Difference in responserate vs placebo, %90% CI

15.00-15.01, 45.01

-9.60-32.54, 13.35

Placebo + AbirateronePatients, n 12 22Responders, n (%)90% CIb

7 (58.3)31.52, 81.59

7 (31.8)18.11, 50.00

CTC, circulating tumor cell.a CTC conversion is defined by a decline to <5 cells/7.5 mL post baseline among patients with CTC ≥5 cells/7.5 mL at baseline.b Patients with a reduction in CTCs of ≥30% compared with baseline were defined as responders for this analysis.

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Table A7. Time to PSA Progression by the Four PTEN Diagnostic Assays Tested Primary Analysis Exploratory Analysis

ICR IHC Ventana IHC FISH NGSPTEN loss

PTEN non-loss

PTEN loss

PTEN non-loss

PTEN loss

PTEN non-loss

PTEN loss

PTEN non-loss

Ipatasertib 400 mg + AbirateronePatients, n 25 32 26 22 28 38 15 21Patients with events, n (%) 18 (72.0) 18 (56.3) 18 (69.2) 12 (54.5) 18 (64.3) 22 (57.9) 8 (53.3) 12 (57.1)

PSA, median months Ipatasertib 3.71 5.59 4.73 5.59 5.55 4.7 5.55 4.7 Placebo 2.79 3.71 1.84 2.83 3.94 2.79 2.79 2.86PSA HR 0.68 0.8 0.55 0.79 0.76 0.68 0.42 0.66 90% CI (0.37-

1.25)(0.48- 1.35)

(0.32- 0.96)

(0.43- 1.46)

(0.42- 1.39)

(0.42- 1.09)

(0.18- 0.97)

(0.33- 1.34)

Ipatasertib 200 mg + AbirateronePatients, n 25 27 31 16 22 47 13 16Patients with events, n (%) 16 (64.0) 21 (77.8) 22 (71.0) 10 (62.5) 16 (72.7) 34 (72.3) 8 (61.5) 11 (68.8)

PSA, median months Ipatasertib 2.92 2.32 2.89 3.78 2.79 2.89 2.92 1.87 Placebo 2.79 3.71 1.84 2.83 3.94 2.79 2.79 2.86PSA HR 0.65 1.61 0.68 0.98 1.17 1.04 0.51 1.14 90% CI (0.35-

1.22)(0.97- 2.68)

(0.40- 1.15)

(0.51- 1.87)

(0.63- 2.16)

(0.69- 1.59)

(0.21- 1.20)

(0.55- 2.35)

Placebo + AbirateronePatients, n 21 35 25 26 21 40 11 13Patients with events, n (%) 14 (66.7) 24 (68.6) 18 (72.0) 18 (69.2) 13 (61.9) 28 (70.0) 8 (72.7) 10 (76.9)

FISH, fluorescence in situ hybridization; HR, hazard ratio; IHC, immunohistochemistry; ICR, The Institute of Cancer Research; NE, not estimable; NGS, next-generation sequencing; PSA, prostate-specific antigen; PTEN, phosphatase and tensin homolog.

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Table A8. Selected AEs by Grouped Terms, Including Those Based on Clinical Experience With Ipatasertib, Preclinical Toxicology Information of Ipatasertib, and AEs Known to Be Associated With Inhibitors of the PI3K-Akt-mTOR Pathway

AE, n (%)

Ipatasertib 400 mg + Abiraterone

(n=84)

Ipatasertib 200 mg + Abiraterone

(n=87)

Placebo +Abiraterone

(n=82)

Diarrhea, any grade 64 (76.2) 40 (46.0) 21 (25.6)

Grade 3 10 (11.9) 2 (2.3) 1 (1.2)

Grade 4 0 0 0

SAE 2 (2.4) 1 (1.1) 0

Asthenia/fatigue, any

grade40 (47.6) 35 (40.2) 35 (42.7)

Grade 3 8 (9.5) 4 (4.6) 3 (3.7)

Grade 4 0 0 0

SAE 1 (1.2) 1 (1.1) 0

Nausea, any grade 44 (52.4) 30 (34.5) 20 (24.4)

Grade 3 2 (2.4) 0 0

Grade 4 0 0 0

SAE 2 (2.4) 0 0

Vomiting, any grade 26 (31.0) 24 (27.6) 12 (14.6)

Grade 3 0 1 (1.1) 0

Grade 4 0 0 0

SAE 0 0 2

Rash, any grade 22 (26.2) 7 (8.0) 7 (8.5)

Grade 3 8 (9.5) 2 (2.3) 0

Grade 4 1 (1.2) 0 0

SAE 2 (2.4) 1 (1.1) 0

Hyperglycemia, any

grade18 (21.4) 8 (9.2) 6 (7.3)

Grade 3 9 (10.7) 2 (2.3) 2 (2.4)

Grade 4 1 (1.2) 0 0

SAE 3 (3.6) 1 (1.1) 0

Hepatotoxicity, any

grade8 (9.5) 6 (6.9) 3 (3.7)

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Grade 3 6 (7.1) 3 (3.4) 2 (2.4)

Grade 4 0 0 0

SAE 0 0 0

Pneumonia, any grade 3 (3.6) 6 (6.9) 2 (2.4)

Grade 3 3 (3.6) 6 (6.9) 0

Grade 4 0 0 0

SAE 3 (3.6) 4 (4.6) 0

Hyperlipidemia, any

grade4 (4.8) 5 (5.7) 0

Grade 3 0 0 0

Grade 4 0 0 0

SAE 0 0 0

Oral mucositis, any

grade5 (6.0) 2 (2.3) 1 (1.2)

Grade 3 0 0 0

Grade 4 0 0 0

SAE 0 0 0

Pneumonitis, any

grade1 (1.2) 1 (1.1) 2 (2.4)

Grade 3 0 0 0

Grade 4 0 0 0

SAE 0 0 0

Colitis, any grade 1 (1.2) 0 1 (1.2)

Grade 3 0 0 0

Grade 4 0 0 0

SAE 0 0 0

AE, adverse event; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; SAE, serious adverse event.Note: No Grade 5 AEs occurred in the AEs listed above.

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