Janice Sung

A Pilot Study of Preoperative Single-Dose Ipilimumab and/or Cryoablation in Women with Early-Stage Breast Cancer with Comprehensive Immune Profiling

Purpose: To assess the safety and tolerability of preoperative cryoablation-mediated tumor antigen presentation and/or ipilimumab-mediated immune modulation in women with operable breast cancer. Experimental Design: In this pilot study, 19 women with breast cancer for whom mastectomy was planned were treated with preoperative tumor cryoablation (n = 7), single-dose ipilimumab at 10 mg/kg (n = 6), or both (n = 6). The primary outcome for this pilot study was safety/tolerability as defined as freedom from delays in pre-planned, curative-intent mastectomy. Exploratory studies of immune activation were performed on peripheral blood and tumor. Results: Preoperative cryoablation and/or ipilimumab were safe and tolerable, with no delays in pre-planned surgery. Grade III toxicity was seen in 1 of 19 (unrelated rash after ipilimumab). Combination therapy was associated with sustained peripheral elevations in: Th1-type cytokines, activated (ICOS+) and proliferating (Ki67+) CD4+ and CD8+ T cells, and posttreatment proliferative T-effector cells relative to T-regulatory cells within tumor. Conclusions: Preoperative cryoablation and single-dose ipilimumab are safe alone or in combination with no surgical delays incurred. Potentially favorable intratumoral and systemic immunologic effects were observed with the combination, suggesting the possibility for induced and synergistic antitumor immunity with this strategy. Clin Cancer Res; 22(23); 5729–37. ©2016 AACR.

Deep Sequencing of T-Cell Receptor DNA as a biomarker of clonally expanded TILs in breast cancer after immunotherapy

Tumor cryoablation plus immune checkpoint blockade facilitates antitumor T-cell responses (TCRs) and improves survival in mice. Deep sequencing of TCRs in human early-stage breast cancer tumors revealed T-cell clonality and density and served as a biomarker after cryo-immunotherapy. In early-stage breast cancer, the degree of tumor-infiltrating lymphocytes (TIL) predicts response to chemotherapy and overall survival. Combination immunotherapy with immune checkpoint antibody plus tumor cryoablation can induce lymphocytic infiltrates and improve survival in mice. We used T-cell receptor (TCR) DNA sequencing to evaluate both the effect of cryoimmunotherapy in humans and the feasibility of TCR sequencing in early-stage breast cancer. In a pilot clinical trial, 18 women with early-stage breast cancer were treated preoperatively with cryoablation, single-dose anti–CTLA-4 (ipilimumab), or cryoablation + ipilimumab. TCRs within serially collected peripheral blood and tumor tissue were sequenced. In baseline tumor tissues, T-cell density as measured by TCR sequencing correlated with TIL scores obtained by hematoxylin and eosin (H&E) staining. However, tumors with little or no lymphocytes by H&E contained up to 3.6 × 106 TCR DNA sequences, highlighting the sensitivity of the ImmunoSEQ platform. In this dataset, ipilimumab increased intratumoral T-cell density over time, whereas cryoablation ± ipilimumab diversified and remodeled the intratumoral T-cell clonal repertoire. Compared with monotherapy, cryoablation plus ipilimumab was associated with numerically greater numbers of peripheral blood and intratumoral T-cell clones expanding robustly following therapy. In conclusion, TCR sequencing correlates with H&E lymphocyte scoring and provides additional information on clonal diversity. These findings support further study of the use of TCR sequencing as a biomarker for T-cell responses to therapy and for the study of cryoimmunotherapy in early-stage breast cancer. Cancer Immunol Res; 4(10); 835–44. ©2016 AACR.

Characteristics and outcome of enhancing foci followed on breast MRI with management implications

AIMnTo evaluate the risk of cancer of an enhancing focus identified at breast magnetic resonance imaging (MRI) by determining the positive predictive value (PPV) associated with specific characteristics of an enhancing focus.nnnMATERIALS AND METHODSnRetrospective, institutional review board-approved review of the database identified 111 consecutive patients who underwent short-term follow-up of 136 enhancing foci in 2008. Kinetic analysis (delayed enhancement pattern) and other characteristics, such as interval change and T2 signal intensity, were evaluated to calculate the PPV for malignancy.nnnRESULTSnThe overall malignancy rate of an enhancing focus was 2.9% [4/136, 95% confidence interval (CI): 0.9-7.6%]. Kinetic analysis showed no statistical difference in PPV between foci with washout enhancement [5.1% (2/39)] versus persistent enhancement [3.2% (2/62); Fishers exact test, pxa0=xa00.6180]. PPV of a T2 hypointense focus was 8.7% (4/46); PPV of a new focus was 13.6% (3/22); PPV of an enlarging focus was 6.7%, (1/15). The combination of a focus being new and T2 hypointense had the highest PPV for malignancy (27.2%, 3/11, 95% CI: 9.2-57.1%).nnnCONCLUSIONnKinetic analysis was not specific for malignancy and should not be used solely to guide management. A new enhancing focus with T2 hypointensity had a high PPV for malignancy and may warrant immediate biopsy.

Abstract P2-15-01: Integrated immunologic assessment of tumor infiltrating lymphocytes (TILs) and peripheral blood to assess synergy of cryoablation (cryo) plus ipilimumab (ipi) in early stage breast cancer (ESBC) patients (pts)

Background: In pts with ESBC, cryo combined with cytotoxic T-lymphocyte antigen 4 blockade was well tolerated and did not delay standard-of-care mastectomy. As observed in mice, cryo+ipi may liberate tumor-associated antigens, synergistically activate tumor-reactive T-cells, and confer long-term anti-tumor immunity. Because singular biomarkers of response to immunotherapy have not been well defined, we conducted an integrated immunologic assessment to explore potential predictors of immune activation and response. Methods: Serial blood and pre-/post-treatment tumor tissue were collected from 18 pts treated with cryo (6 pts), single-dose ipi at 10mg/kg (6 pts), or cryo + ipi (6 pts). A Meso Scale Discovery platform was used to measure plasma cytokine interferon gamma (IFNγ). Multiparameter flow cytometry was used to evaluate peripheral and intratumoral T-cell and myeloid cell quantity, T-cell phenotype (effector versus regulatory), proliferation state (Ki67), and activation state (inducible costimulator [ICOS] expression). Finally, a DNA deep sequencing platform was used to conduct T-cell repertoire analysis of peripheral T-cells and TILs. Results: Sustained >2-fold elevations (1 month post-treatment) in plasma IFNγ were observed in the majority (4/6) of pts receiving cryo/ipi (median 6-fold increase), but in the minority of pts receiving cryo (0/6, median 0-fold) or ipi (2/6, median 0-fold). Similarly, sustained >2 fold elevations in ICOS expression in peripheral CD3+CD4+ T-cells, a known pharmacodynamic marker of ipi, were observed in the majority (5/6) of pts receiving cryo/ipi (median 4-fold increase), but in the minority of pts receiving cryo (0/6, median 0-fold) or ipi (2/6 ipi; median 1-fold). No trends were observed in peripheral myeloid derived suppressor cells. Analysis of TILs by flow cytometry identified increased numbers of proliferating CD8+ T-cells (CD8+Ki67+) in ipi and cryo/ipi groups relative to cryo alone; furthermore, the ratio of proliferating (CD8+Ki67+) to regulatory (CD4+CD25+FoxP3+) cells was enhanced in the cryo/ipi group. Finally, analysis of T-cell repertoire in TILs demonstrated that cryo/ipi generated an influx of novel T-cell clones, with select clones surging dramatically in predominance and circulating within the periphery. Conclusions: Utilizing an integrated assessment, we identified evidence of immunologic synergy with combination cryo/ipi versus either therapy alone. Of the tested parameters, peripheral CD4+ ICOS expression, plasma IFNγ, Ki67-gated TIL effector/regulatory ratios, and clonal repertoire analysis were identified as promising biomarkers of immune activation. These findings will inform a prospective assessment of potential immunologic biomarkers of immune response and clinical benefit in a phase 2 study of cryo-immunotherapy in ESBC. Citation Format: David Page, Jianda Yuan, Adi Diab, Zhiwan Dong, Arielle Ginsberg, Phillip Wong, Ryan Emerson, David Redmond, Brian Blum, Zhenyu Mu, Chunjun Zhao, Christopher Comstock, Elizabeth Morris, Elizabeth Comen, Alan Kotin, Janice Sung, Edi Brogi, Monica Morrow, Stephen Solomon, Virgilio Sacchini, Majid Maybody, Deirdre Neville, Harlan Robins, Sujata Patil, Jedd Wolchok, Clifford Hudis, Larry Norton, James Allison, Padmanee Sharma, Heather McArthur. Integrated immunologic assessment of tumor infiltrating lymphocytes (TILs) and peripheral blood to assess synergy of cryoablation (cryo) plus ipilimumab (ipi) in early stage breast cancer (ESBC) patients (pts) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-15-01.

Abstract OT3-1-01: A pilot study of single-dose ipilimumab and/or cryoablation in women with early-stage breast cancer scheduled for mastectomy

Background: With cryoablation, probes are inserted into tumors to induce crystallization, osmotic changes, vascular stasis, and ultimately tumor necrosis. The resultant release of tumor antigens could activate a tumor-specific immune response through antigen presentation by dendritic cells (DC) to T-cells. Cryoablation is an effective treatment modality in many cancers (Habash 2007), and feasibility studies have shown that thermal ablation can be performed safely and effectively in small breast cancers (Noguchi 2007). To amplify immune response, we use ipilimumab, a fully human monoclonal antibody that blocks cytotoxic T lymphocyte antigen-4 (CTLA4), a co-inhibitory molecule present on the surface of activated T cells. Durable and clinically significant responses to ipilimumab have been reported in prostate, bladder, and renal cell cancers. Ipilimumab has been most extensively studied in malignant melanoma, with 10–20% response rates and significant survival benefits reported (Wolchok 2010, Hodi 2010). Cryoablation-mediated tumor destruction exposes DCs to sufficient quantities of tumor antigens and inflammatory cytokines to induce DC maturation and activation. Furthermore, in preclinical murine models, the combination of cryoablation with CTLA4 blockade successfully mediates rejection of metastatic prostate cancer lesions and prevents growth of secondary tumors (Waitz 2012). Combined ablative therapy with CTLA4 blockade is appealing due to the relative safety of percutaneous ablation in early stage breast cancer and the efficacy and tolerability of ipilimumab administration in numerous settings. We therefore hypothesize that this strategy could confer long-term immunity, and ultimately cure, for women with early stage breast cancer. In this pilot study, the safety and tolerability of pre-operative cryoablation alone, single dose ipilimumab (10mg/kg) alone, or the combination is being evaluated in women with early stage breast cancer. Design: Three sequential test groups of 6 patients are being enrolled (total N=18). The study groups are: pre-operative cryoablation alone (A), pre-operative single dose ipilimumab alone (B), and pre-operative single-dose ipilimumab followed by cryoablation (C). Groups A and B must meet the safety primary endpoint before enrollment to study arm C is begun. The timing of all study interventions is defined by the pre-determined, non-study surgery date. Primary aim: To determine the safety and tolerability of pre-operative ipilimumab and/or cryoablation in patients with resectable breast cancer. Secondary aims: To explore and characterize pre- and post-intervention radiographic and immunological (peripheral blood and tumor tissue) correlates. Statistics: If at least 5 of the 6 patients in each group proceed with surgery without delay, the regimen will be considered safe/tolerable for further study. Secondary aims are exploratory. Eligibility: Eligible women are age ≥18y with early-stage, operable, ≥1.5 cm invasive breast cancer, no history of autoimmune disease and planning mastectomy. Study Status: As of June 12, 2012, Study Arm A is filled: 4 women have undergone cryoablation and mastectomy and 2 have undergone cryoablation and are awaiting surgery. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr OT3-1-01.

Matched T cell repertoire analysis of peripheral blood and tumor-infiltrating lymphocytes (TILs) in early stage breast cancer (ESBC) patients (pts) treated with pre-operative cryoablation (cryo) and/or Ipilimumab (Ipi)

Meeting abstractsnnCryo plus anti-CTLA-4 therapy induces antigen-specific clonal T cell expansion, enhanced survival, and long-term anti-tumor immunity in mice [[1][1]]. We recently demonstrated that pre-operative cryo and/or anti-CTLA-4 therapy with Ipi is well tolerated and clinically feasible in

Abstract P4-13-01: A pilot study of pre-operative (Pre-op), single-dose ipilimumab (Ipi) and/or cryoablation (Cryo) in women (pts) with early stage/resectable breast cancer (ESBC)

Background : Intratumoral cryo combined with immune modulation generates a potent systemic anti-tumor immune response that might improve recurrence free survival in ESBC. In this study, we evaluate the safety of pre-op cryo and/or ipi (10mg/kg) in pts with ESBC. Radiographic correlates and intratumoral/serologic immune responses are also explored. Methods : Eligible pts are ≥18y of age with operable ≥1.5 cm invasive ESBC, no history of autoimmune disease and planned mastectomy. Pts are sequentially assigned to receive pre-op: cryo alone (group-A), ipi alone (B), or ipi with cryo (C). Cryo is performed 7-10d prior to surgery. Ipi is administered 8-15d prior to surgery (1-5d prior to cryo). If at least 5/6 pts in each group proceed with surgery without delay, the regimen will be considered safe/tolerable. Toxicity evaluation continues for 12 wks after ipi administration for groups B and C. Cytokine levels are measured from peripheral blood samples at multiple time points [baseline, at biopsy/cryo (post ipi), at mastectomy, and 30 days post surgery] using MSD® cytokine assay. Results: As of May 30, 2013, 7/7 pts were enrolled to group A (expanded after a possible technical failure in 1 pt) and 6/6 pts were enrolled to group B. The median age was 45y (range 39-69y). All 13 pts in groups A and B underwent mastectomy without delay. Group C is now accruing with 1/6 patients enrolled and surgery completed without delay. Toxicity and safety: 6/7 pts in group A and 0/6 in group B had ischemic tumor necrosis/infarction in the mastectomy tissue. Overall, pre-op cryo or ipi alone have been well tolerated with no study related grade ¾ adverse events (AE) reported (Table1). Exploratory studies are ongoing for pts who successfully complete the planned intervention. Currently, immune response data is available for 10 pts: 5 from each of groups A and B. Both groups have detectable IL-8 levels which are increased from baseline, interestingly and only in group-B (ipi alone) several pts have preferential increase of IFN-γ and TNF-α (TH1 cytokines) compared to IL-5 and IL-10 (TH2 cytokines). One group B pt who had pre-treatment peri-tumoral lymphocytic infiltrate had prolonged and significantly higher levels of IFN-γ and TNF-α only. Conclusion : To date, pre-op cryo or ipi is safe and tolerable in pts with ESBC. Preliminary exploratory analyses from groups A and B demonstrated elevated IFN-γ and TNF-α levels in the ipi-treated group. A Phase II study of pre-op ipi and cryo in ESBC is planned. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-13-01.

P2-08-01: Impact of Aromatase Inhibitors on Background Parenchymal Enhancement and Amount of Fibroglandular Tissue on Breast MRI.

Background: On breast MRI, background parenchymal enhancement (BPE) and volume of fibroglandular tissue (FGT) have been shown to reflect a patient9s hormonal status. Tamoxifen has been shown to reduce mammographic breast density and may serve as an early predictor of response in the prevention setting (Cuzick, JNCI 2011). We have shown that adjuvant tamoxifen can reduce BPE in the unaffected breast in women with breast cancer. We hypothesize that aromatase inhibitor (AI) induced endocrine changes in breast tissue should also be evident and therefore we performed a study to evaluate whether adjuvant AI therapy influences BPE or amount of FGT in the contralateral breast. Methods: An electronic medical record review identified 856 postmenopausal women with stage I-III breast cancer who had at least two breast MRIs and took adjuvant AI treatment. A retrospective chart review was conducted to select those patients without a history of prior tamoxifen or raloxifene treatment who had a MRI of the contralateral breast both before and during 6 to 12 months of AI treatment. After exclusion of all irradiated breasts, 168 women were eligible. MRIs were performed between August 1999 and June 2010. Two radiologists who were blind to AI treatment status, independently rated level of BPE and amount of FGT using categorical scales: BPE — Minimal, Mild, Moderate, Marked; FGT — Fatty, Scattered, Heterogeneously Dense, Dense (based on proposed BIRADS criteria for BPE and on ACR criteria for FGT). Blinded side-by-side direct comparison evaluated whether there was a category change between the two MRIs. A consensus was reached in cases of disagreement. The Wilcoxon signed-rank test was used to assess changes in rating categories for BPE and FGT between before and during AI breast MRIs. A waiver of authorization was granted by the institutional review board for this study. Results: In this study 127/168 (76%) women were treated with anastrozole, 33/168 (20%) with letrozole and 8/168 (5%) with exemestane. Based on the blinded side-by-side comparison, a category (or more) decrease in BPE occurred during treatment with AIs (p Conclusions: After 6 to 12 months of treatment with adjuvant AIs, there was a statistically significant category (or more) decrease in BPE. BPE is more sensitive than FGT to changes in normal breast stroma that occur during adjuvant treatment with AIs and BPE may be a marker of anti-hormonal activity in the breasts. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-08-01.