Abstract P2-04-07: Immune profiling of post neoadjuvant high metastatic risk (RCB-II/III) residual disease in patients with early triple negative breast cancers
Authors: Irshad S , Cheang M , Gazinka P , Naidoo K , Buus R , Pinder S , Dowsett M , Tutt A 
Published: March 2017

Abstract
Background: Poor prognosis in TNBC can be predicted in the significant fraction of patients with large volume residual cancer burden (RCB-II/III) after neoadjuvant chemotherapy (NACT). Whilst residual disease has been characterised to identify “driver” mutations and copy number variations, the contribution of the immune response within its tumour microenvironment remains unclear. Here we aimed to: 1) assess the potential spatial heterogeneity of immune transcript related gene expression between areas of tumour approximately 1cm apart as might still occur with a radiologically guided biopsy through the residual disease; and 2) assess the immune stroma composition of the TNBC high metastatic risk RCB II/III disease.
Method: 12 TNBC post NACT RCB II/III residual cases were identified from the KHP biobank. H&E sections were reviewed and areas of tumor 1cm apart within a residual resection specimen marked as area A and area B. HistoQuest analysis software was used to quantify the proportion of tumor infiltrating lymphocytes (per total cell count) within both areas. RNA was extracted from both areas and immune gene expression profiling performed using a Nanostring nCounter® on all 24 samples. The immune PanCancer panel consisted of 770 genes combining markers for different immune cell populations. Differential genes between paired samples were compared and unsupervised hierarchical clustering using 770 genes and immune cell types performed.
Results:Quantitative comparison of the tumour infiltrating lymphocytes (TILS) between area A and B revealed that 73% (8/11) of the cases had a &lt;2-fold difference in the percentage of TILS within a residual specimen; and 27% (3/11) displayed a &gt;2-fold (range 2.03-3.16) difference in the TILS. When comparing the 770 gene expression profiles between sampling areas in the same tumour, we found little spatial heterogeneity with areas A/B clustering together in 10 out of 12 cases. Interestingly, the two cases that revealed spatial heterogeneity within the paired samples displayed little immune cell heterogeneity histologically (i.e. &lt;1-fold change in the TILS percentage score between area A and B). Comparing patient samples by immune gene expression profiling divided the patients into two groups: i) those with immunologically enriched tumors in whom gene signatures for majority of the immune cell types (DC, Macrophages, CD8+T-cells, T-helper cells (Th17, Th2, Th1), Tregs, NK, B-cells, Neutrophils, Mast cells) were highly expressed and ii) those with immunologically inert tumors in whom the immune cell signatures were not highly expressed. Within our patient cohort, patients with immunologically enriched gene expression profiles were also seen to display higher TILS score (ranging between 28.17% to 40.66%) as compared to patients with immunologically inert gene expression with scores ranging from 11.82% to 16.80%.
Conclusion: The findings that high metastatic risk residual disease can be further characterized as either “immunologically inert” or “immunologically enriched” at the level of extensive immunological transcript gene expression and by histological assessment of TILS requires further investigation; and is being validated in a larger sample set.
Citation Format: Irshad S, Cheang M, Gazinka P, Naidoo K, Buus R, Pinder S, Dowsett M, Tutt A. Immune profiling of post neoadjuvant high metastatic risk (RCB-II/III) residual disease in patients with early triple negative breast cancers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-07.