Oncolytic virotherapy for treatment of breast cancer, including triple-negative breast cancer

Figures & data

Figure 1. Combination of Ad5/3-D24-GMCSF with 4-hydroperoxycyclophosphamide (4-HP-CP) or cyclophosphamide (CP) increases cell killing of MDA-MB-436 TNBC cells in vitro. Viability of MDA-MB-436 TNBC cells after infection with (A) Ad5/3-D24-GMCSF + CP or (B) Ad5/3-D24-GMCSF + 4-HP-CP. Cell viability was measured 5 d after infection by MTS assay. Viability of mock-infected cells was set as 100%. Means with standard deviations of triplicates are shown. VP, viral particles; CP, cyclophosphamide; 4-HP-CP, 4-hydroperoxycyclophosphamide. (A) * p < 0.05 Virus + 4-HP-CP vs. 4-HP-CP alone; ** p < 0.01 Virus + 4-HP-CP vs. virus alone; *** p < 0.001 Virus + 4-HP-CP vs. virus alone. (B) ** p < 0.01 Virus (1 VP/cell) + CP vs. virus alone; Virus (100 VP/cell and 1,000 VP/cell respectively) vs. CP alone.

Figure 2. Combination of Ad5/3-D24-GMCSF with low-dose CP displays enhanced antitumor efficacy in a TNBC mouse model. Nude/NMRI mice were inoculated orthotopically into two different mammary fat pad sites with human TNBC cells (MDA-MB-436). When tumors reached the size of approximately 5 mm diameter, they were injected with 7 × 10⁹ VP/tumor of Ad5/3-D24-GMCSF or saline on days 1, 4, 8, 15, 29, 43. Low-dose CP (20 mg/kg) was administered to indicated groups intraperitoneally. Tumor sizes are indicated as percentage respective to day 1, which was set as 100%. Data is presented as means + standard deviations. ** p < 0.01 Ad5/3-D24-GMCSF + CP vs. Ad5/3-D24-GMCSF alone on day 33 post-infection. Tumor sizes were compared between the treatment groups using Mann-Whitney U test with Bonferroni correction. n = 8 tumors/group; n = 10 tumors/mock groups.

Figure 3. Waterfall plots showing radiological and tumor marker responses after Ad5/3-D24-GMCSF treatments in breast cancer patients. (A,B) Percent change in tumor burden relative to pre-treatment baseline is represented. (A) CT, and (B) PET-CT are shown. (C) Percent change in Ca15–3 or CEA levels (patients R170, R249 and R328) relative to pre-treatment baseline is represented. Patient R196 (PD) is not represented in the graph (percent change was not measured). For patient R170, “best response” is shown. TNBC patients are underlined. Mets, metastases. (A,B) n = 13. Patients R247 and R248 have been imaged with both CT and PET-CT; (C) n = 12.

Table 1. Clinical responses and patient survival. Patients R184, R195, R317, R328 had triple negative breast cancer.

Patient code	Number of treatments with Ad5/3-D24-GMCSF	Concomitant CP	Imaging result	Tumor markers	Survival (days)^1
R8	1	Yes	NE^2	Ca15–3: MR (−16%)	717
R73	2	Yes	SD^3		896
		Yes	SD (+2%)^4,^5	Ca15–3: MR (−29%)
R161	1	Yes		Ca15–3: PD (+34%)	137
R170	1	Yes	PD (+60%)^4,^6	CEA: MR (−10%) Ca15–3: PD (+129%)	107
R172	1	No			112
R184	3	Yes	PD (+21%)^4		227
R195	1	No	PD (+33%)^4,^6		241
R196	1	No	PD (+47%)^4,^6	Ca15–3: PD	162
R203	1	Yes	SD (+4%)^4,^6	Ca15–3: MR (−18%)	442
R247	3	Yes	PET-CT: PMR (−38%)^4 CT: MR (−10%)^4	Ca15–3: PR (−30%)	195
R248	3	Yes	PET-CT: SMD (+19%)^4 CT: PD (+45% +new mets)	Ca15–3: PD (+56%)	393
R249	3	Yes	PET-CT: PMD (+36%)^4	CEA: MR (−24%)	497
R255	3	Yes	PET-CT: PMD (+56%)^4	Ca15–3: PD (+29%)	1771^7
R256	3	Yes	PET-CT: PMD (+70%)^4 CT: PD (new bone mets)	Ca15–3: SD (−5%)	189
R317	3	Yes	PET-CT: PMD (+54%+new mets)^4	Ca15–3: PD (+74%)	190
R328	3	Yes	PET-CT: PMD (–22% in injected lesions + two new mets)^4	CEA: CR (–70%)	239

¹ Numbers indicate the survival from their 1^st Ad5/3-D24-GMCSF treatment

² Patient not evaluable according to modified RECIST 1.1. Patient showed a complete response (CR) after two additional virus treatments prior to Ad5/3-D24-GMCSF and remained in CR when imaged 7.5 mo after Ad5/3-D24-GMCSF treatment.

³ Patient received one additional virus treatment prior to Ad5/3-D24-GMCSF. Imaging was performed 9.5 weeks after Ad5/3-D24-GMCSF treatment

⁴ Serial treatment, imaging was performed after the third treatment

⁵ Patient received one additional virus treatment prior to Ad5/3-D24-GMCSF and one after it. Imaging was performed after the third treatment, 6 weeks after Ad5/3-D24-GMCSF injection. New CT 15 weeks after Ad5/3-D24-GMCSF injection: PD (+1.4% + new liver metastasis + a few new lymph nodes in the peritoneum)

⁶ Patient received two additional virus treatments after Ad5/3-D24-GMCSF. Imaging was performed after the third treatment.

⁷ Patient alive at the end of follow-up (March 2015)

Abbreviations: NE, not evaluable; CP, cyclophosphamide; CT, contrast-enhanced computed tomography; PET-CT, [18F]-fluorodeoxyglucose positron emission tomography; PD, progressive disease; SD, stable disease; MR, minor response; PMD, progressive metabolic disease (in PET); SMD, stable metabolic disease; mets, metastases. Percentage change in tumor diameters and appearance of new lesions indicated for CT and percentage change in summed SUVmax, as well as appearance of new lesions for PET-CT.