Background: APT is a single arm multicenter, phase II study of paclitaxel and trastuzumab. Patients with HER2+ breast cancer with negative nodes and tumor size < 3 cm were eligible. Disease-free survival at 7 years was 93.3% with only 4 distant recurrences. Characterizing intrinsic subtype and immune profiles of these smaller tumors may help us better understand if the biology of smaller HER2+ tumors is different than larger tumors that have been previously characterized. Methods: Intrinsic subtyping by PAM50 and immune signatures by PanCancer Immune Profiling Panel were performed on the nCounter Analysis system on archival tissue. Tissue was also tested by immunohistochemistry for PDL1 (tumoral and immune cells) and was assessed for tumor infiltrating lymphocytes (TILs) using guidelines from the International TILs Working Group. TILS were categorized as follows: low (≤10%), intermediate (10-60%), high (≥60%); PD-L1 was characterized as low (0-1%), intermediate (1-10%), and high (>10%). Results: PAM50 data were available for 209 of the 406 cases: 142 (68%) were HER2-Enriched (HER2E), 22 (11%) Luminal A, 25 (12%) Luminal B, and 20 (10%) Basal-like. Immune profile information was available for 162 cases, of which 138 also had intrinsic subtype data. 184 cases were evaluated for PD-L1, and 210 cases for TILs. There was a strong correlation between PD-L1 in the tumor and lymphoid compartments (McNemar's chi-squared, p= 6.5x10-12). High tumoral PD-L1 was seen with higher frequency in the HER2E subset, while high immune cell PD-L1 and high TILs were seen with higher frequency within both the HER2E and Basal-like subtypes (Table). Immune profile data demonstrates that the T-cell signature has the strongest association with TILs, and that luminal A tumors tend to have very similar immune profiles while there is more variable expression of immune cell types with the basal-like tumors. Further work is ongoing to complete PAM50 and intrinsic subtype profiling of remaining tumors, and additional relationships between immune profile and intrinsic subtype will be presented at the meeting. Conclusions: The majority of small HER2+ breast cancers are HER2E, and tumors of this intrinsic subtype have the highest prevalence of high expression of PDL1 and high TILs, suggesting that these tumors may be more immunogenic than the luminal A and B HER2+ tumors. Further work to explore immune profile data by intrinsic subtype is ongoing and may have implications for future trial design. HER2ELuminal ALuminal BBasal-LikePDL1 (tumoral) Low6712159Intermediate16124High8100PDL1 (immune cell) Low3712113Intermediate27154High26116TILs Low6415165Intermediate36308High7011 Citation Format: Tolaney SM, Barry W, Guo H, Dillon D, Tan YB, Fuhrman K, Osmani W, Getz A, Baltay M, Dang C, Yardley D, Moy B, Marcom K, Krop I, Winer E. Immune profile of small HER2+ tumors in the APT trial [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD3-01.
The presence in plasma of mitochondrial DNA (mtDNA) fragments, a proinflammatory damage-associated molecular pattern (DAMP), is positively associated with outcomes of multiple human disorders. Because mtDNA comprises only a small percentage of the total DNA in plasma, qPCR is typically employed as an analytic strategy. However, this method provides little insight into sequence origins or other characteristics of circulating mtDNA fragments. Here we found that target bait-capture applied to plasma mtDNA derived from severely injured patients enriched recovery by ≈ 1400-fold, thus affording depth sufficient for NextGen sequence analysis. Our method excluded nuclear mitochondrial insertions (NUMTs) using a stringent alignment and filtering strategy which calls and quantifies both NUMT in the reference genome and polymorphic NUMTs. After enrichment, we sequenced 2,000–60,000x mean coverage over the mtDNA genome. Normalization of mtDNA abundance to NUMT coverage reduced batch variability. Two massively-transfused trauma patients and two non-transfused patients displayed time-dependent increases in mtDNA DAMP coverage and decreases in the mean fragment length, which were more pronounced in massively transfused patients. Finally, our approach enabled detection of low-frequency heteroplasmic variants. Collectively, these findings suggest that our target bait-capture, deep sequencing and attendant analytic protocols could provide unprecedented characterization of cell-free plasma mtDNA and NUMTs.
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