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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)

  • David B Page1,
  • Heather McArthur1,
  • Zhiwan Dong2,
  • Phillip Wong1,
  • Ryan Emerson3,
  • Zhenyu Mu2,
  • Chunjun Zhao2,
  • Christopher Comstock1,
  • Elizabeth Morris1,
  • Elizabeth Comen1,
  • Alan Kotin1,
  • Janice Sung1,
  • Edi Brogi1,
  • Monica Morrow1,
  • Stephen Solomon1,
  • Virgilio Sacchini1,
  • Majid Maybody1,
  • Deirdre Neville1,
  • Adi Diab4,
  • Padmanee Sharma4,
  • Harlan Robins5,
  • Sujata Patil1,
  • Jedd D Wolchok1,
  • Clifford Hudis1,
  • Larry Norton1,
  • James Allison4 and
  • Jianda Yuan6
Journal for ImmunoTherapy of Cancer20142(Suppl 3):P138

Published: 6 November 2014


Peripheral Blood Mononuclear CellCell CloneIpilimumabDeep SequencingEarly Stage Breast Cancer


Cryo plus anti-CTLA-4 therapy induces antigen-specific clonal T cell expansion, enhanced survival, and long-term anti-tumor immunity in mice [1]. We recently demonstrated that pre-operative cryo and/or anti-CTLA-4 therapy with Ipi is well tolerated and clinically feasible in women with ESBC. Furthermore, cryo with or without Ipi generates a polyclonal influx of novel T cell clones within the tumor bed [2, 3]. Here, we utilize T cell repertoire analysis to explore the impact of cryo and/or Ipi on clonal expansion within peripheral blood and TILs.


In a pilot study, women with ESBC were treated with cryo 7-10d before mastectomy (6 pts), single-dose Ipi (10 mg/kg) 8-15d before mastectomy (6 pts), or cryo+Ipi (6 pts). Peripheral blood mononuclear cells (PBMCs) and tumor tissue were obtained pre-mastectomy (immediately preceding cryo and/or 1-5d after Ipi), and at mastectomy. T cell repertoire analysis was conducted on extracted DNA using an Illumina® DNA deep sequencing platform and ImmunoSEQ™ software. Clones comprising ≥0.01% of sample DNA were analyzed, and results are reported descriptively.


Cryo with or without Ipi was associated with decreases in absolute TIL count (median change: Ipi +6%, cryo -73%, cryo+Ipi -16%). However, cryo+Ipi was associated with the greatest expansion of TIL clones across the range of 102-104 amplicons (table 1), although no difference was observed by group in PBMC clones. Across all samples, a median of 523 TIL clones increased by ≥102 amplicons, and a median of 4 TIL clones increased by ≥103 amplicons. The Ipi/cryo group exceeded the median in 80% (4/5) of cases. 21% of all TIL clones were detectable in time-matched PBMC, whereas 16% of expanding (≥102) TIL clones were detectable in time-matched PBMC.
Table 1

Therapy-associated T cell clonal expansion in TILs and PBMCs.


Median # TIL clones expanding by

Median # PBMC clones expanding by


≥102 amplicons

≥103 amplicons

≥104 amplicons

≥102 amplicons

≥103 amplicons

≥104 amplicons























Cryo plus Ipi expands more TIL clones than either strategy alone. Therapy-associated clonal expansion may be difficult to detect in PBMCs. These data highlight the potential importance of TIL repertoire analysis for the monitoring of pts treated with cryo and/or Ipi in the preoperative setting. In a follow-up randomized study, we will evaluate whether TIL clonal expansion across the 102-104 range can be used to predict recurrence-free survival.

Authors’ Affiliations

Memorial Sloan Kettering Cancer Center, New York, USA
Sloan Kettering Institute, New York, USA
Adative Biotechnologies, Seattle, USA
MD Anderson Cancer Center, Houston, USA
Fred Hutchinson Cancer Center, Seattle, USA
Immune Monitoring Facility, Sloan Kettering Institute, New York, USA


  1. Waitz R, Solomon S: Potent induction of tumor immunity by combining tumor cryoablation with anti-CTLA-4 therapy. Cancer Res. 72: 430-9.Google Scholar
  2. Diab A, McArthur H: A pilot study of preoperative (Pre-op), single-dose Ipilimumab (Ipi) and/or cryoablation (Cryo) in women (pts) with early-stage/resectable breast cancer (ESBC). ASCO Annual Meeting. 2014, Abs #1098, Chicago, ILGoogle Scholar
  3. Page D, Yuan J: T cell receptor (TCR) DNA deep sequencing to evaluate clonality of tumor-infiltrating lymphocytes (TILs) in early-stage breast cancer patients (pts) receiving preoperative cryoablation (cryo) and/or Ipilimumab (Ipi). ASCO Annual Meeting. 2014, Chicago, IL, , Abs #3021Google Scholar


© Page et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.