Abstract P5-04-22: Immune responses triggered by cryoablation of breast cancers
Authors: Campoverde Allison A , Klein Jonah D , Aukers Zachary , Kennedy John , Zemba-Palko Vlasta , Ciocca Vincenzo , Ciocca Robin M , Sabol Jennifer L , Carp Ned Z , Wallon Margaretha 
Published: March 2020

Abstract
Background: Cryoablation, the destruction of cells by ultra-low temperatures, has been used to treat benign breast disease and two clinical trials (ACOSOG Z1072 and FROST) have been conducted to determine its utility in invasive breast cancers. The focus of the trials has been the rate of complete tumor ablation with no assessment of immunological responses.
We hypothesize that neoantigens released during cryoablation might be sufficient to trigger a robust immune response to prevent and/or reduce spread and relapse of breast cancers.
In this pilot study we 1) evaluated biopsy material, cryoablated specimens and axillary lymph nodes from patients with cancers smaller than 2cm enrolled in the ACOSOG Z1072 trial at Lankenau Medical Center [N=18] and 2) assessed immune responses and effects on metastases formation in the classical mouse mammary tumor model 4T1 in immune competent Balb/c mice. In both settings responses were compared to patients/mice treated with surgical resection alone.
Methods: After obtaining IRB approval for retrospective analyses of specimens from the ACOSOG Z1072 trial, immunohistochemical staining of surgical specimens was performed. Sections were stained for CD4, CD8, CD20, CD21, and CD1c. In the IACUC approved animal experiments, 4T1 cells were injected orthotopically in the mammary fatpad to initiate tumor growth. Small tumors were treated by cryoablation or surgery alone. Animals were euthanized 7 days post-treatment and tissues were collected to assess cytokine levels and presence of dissociated 4T1 cells. Single-cell suspensions of tumor, tumor-draining lymph node [TDLN], and spleen were tested for secretion of mouse Th1/Th2 cytokines using a bead array and measured by flow cytometry. Possible metastatic spread was assessed by a clonogenic assay using cells from venous blood, lung and brain. Cell suspensions were seeded in growth medium with the selection agent 6-thioguanine, allowing only resistant 4T1 cells to form colonies.
Results: Cryoablation transformed tumors in both patients and mice into a gelatinous mass surrounded by a fibrotic capsule. Sections of tumors from both humans and mice displayed a necrotic core and infiltrating lymphocytes in the microenvironment. The cryoablated human tumors had slightly higher presence of lymphocytes positive for CD8+ compared to CD4+. The inverse relation was observed in non-cryoblated specimens. No significant difference was observed for CD20+ lymphocytes. Tumor-draining lymph nodes from cryoablated patients had an elevated presence of CD20+ B cells compared to patients treated by surgery alone. Follicular dendritic cells (CD21+) were also present at higher numbers in TDLN from cryoablated patients. Animals treated with cryoablation displayed robust increases of Th1 and Th2 cytokines in both spleen and TDLN compared to animals with surgery treatment. In the animals, circulating tumor cells were found prior to treatment, while no 4T1 colonies formed from cell suspensions of lung and brain tissue [N=8]. At end-point, the surgery group had more 4T1 foci formed from lung and brain [mean foci /animal = 6.25 and 0.75, respectively; N=6] than the cryo group that had 2.25 and 0 foci in lung and brain, respectively [N=8].
Conclusion: Cryoablation of breast cancer lesions can induce stimulatory immune responses in vivo. These immune responses might explain why animals treated with cryoablation, though having circulating tumor cells at the time of treatment, exhibited fewer micro metastatic growths compared to surgery alone. The presence of elevated numbers of CD20+ in TDLN has been associated with improved disease-free survival. All local patients in the clinical trial are currently disease-free (5 to 9-year F/U) which is higher than expected recurrence rate at ~15% at 9 years post treatment.
Citation Format: Allison A Campoverde, Jonah D Klein, Zachary Aukers, John Kennedy, Vlasta Zemba-Palko, Vincenzo Ciocca, Robin M Ciocca, Jennifer L Sabol, Ned Z Carp, Margaretha Wallon. Immune responses triggered by cryoablation of breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-22.