Background: Breast cancers are traditionally viewed as immunologically silent. However, recent research has demonstrated that the triple-negative breast cancer (TNBC) and HER-2 positive (HER2) are capable of stimulating the immune system. In contrast, the most commonly diagnosed subtype, the luminal A (hormone receptor positive (HR+)/HER-2 negative), is associated with a lower degree of tumor infiltrating lymphocytes (TILs) and low expression level of immune checkpoint regulators such as programmed death-ligand-1 (PD-L1). Cryoablation, the destruction of cells by ultra-low temperatures, has been used to treat benign breast disease and clinical trials have been conducted to determine its utility for invasive breast cancers. The primary endpoint has been the rate of complete tumor ablation with no assessment of immunological responses. We hypothesized that neoantigens released during cryoablation might be sufficient to trigger immune responses that could aid in the prevention/reduction of metastatic spread and relapse of breast cancers.In this pilot study we evaluated biopsy material, cryoablated specimens and sentinel lymph nodes (SLN) from patients enrolled in the ACOSOG Z1072 trial at Lankenau Medical Center (LMC) [N=18] with cancers < 2 cm. Responses were compared to patients treated with surgical resection.Methods: After obtaining IRB approval for retrospective analyses of specimens from the ACOSOG Z1072 trial, immunohistochemical staining of archival specimens was performed. An on-going biomarker study protocol was amended to include immune markers and used to select control patients of similar age and tumor characteristics. Per trial protocol, tumors had to be be surgically removed within 28 days of cryoablation, but the exact time was left to the surgeons discretion. Participating surgeons at LMC preferred to remove tumors on average 24.7 days after ablation resulting in an average time from diagnosis to surgical removal of 43.3 days (range 25- 56 days). Although most breast surgeries are performed within two weeks of diagnosis; some patients defer their surgery for personal reasons. This allowed us to identify patients (N=26) with a comparable average time from diagnosis to surgery of 37 days (range 21 - 66 days). Sections were stained for CD4, CD8, CD20, CD21, and CD1c to assess possible changes in immune repertoire due to cryoablation. We also evaluated presence of immune check-point regulators such as PD-L1 and CTLA-4 in biopsy, surgical material and SLN together with the immune modulator IDO1 using commercially available antibodies and standard techniques. Results: Cryoablation transformed tumors in patients into a gelatinous mass surrounded by a fibrotic capsule. The ACOSOG trial demonstrated an effective cryoablation-induced destruction of ER+ invasive ductal carcinoma lesions in 92% of patients (N=86). Sections from cryoablation patients displayed a necrotic core and infiltrating lymphocytes in the microenvironment. These masses had a slightly higher presence of CD8+ lymphocytes compared to CD4+. The inverse relation was observed in non-cryoblated specimens. SLNs from cryoablated patients had an elevated presence of CD20+ B cells compared to patients treated by surgery. Follicular dendritic cells (CD21+) were also present at higher numbers in SLNs from cryoablated patients. We detected positive staining for both PD-L1 and IDO1 among surgical and cryoablated patients. Sporadic presence of CTLA-4 was identified with a slight preference for cryoabalted patients. Conclusion: Cryoablation of breast cancer lesions can induce immune responses in vivo with possible anti-tumor activity in immunologically silent tumors such as HR+ breast cancers. All local cryo patients in the clinical trial are currently disease-free (F/U 5 - 13years), while 1 surgical patient is alive with late recurrence (11 years post-surgery).

Citation Format: Margaretha Wallon, Jonah D Klein, Zachary Aukers, Nolan Metz, Vibha Ahudja, Laura Mandik-Nayak, Vincenzo Ciocca, Vlasta Zemba-Palko, Robin M Ciocca, Jennifer L Sabol, Ned Z Carp. Development of unique immune responses triggered by cryoablation of breast cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-04-05.