Abstract 4899: Tumor T-cell receptor (TCR) diversity elucidates the immune response to genetic alterations of muscle-invasive bladder cancer
Authors: Choudhury Noura , Yap Kai Lee , Kiyotani Kazuma , Yew Poh Yin , Campanile Alexa , Antic Tatjana , Steinberg Gary , Park Jae-Hyun , O'Donnell Peter H. , Nakamura Yusuke 
Published: August 2015

Background: Muscle-invasive bladder cancer (MIBC), unlike the more favorable low-grade papillary form, has a dismal prognosis with a significant risk of recurrence after surgical resection. Previous whole-exome sequencing of MIBC tumors performed by our lab demonstrated that carriers of mutations in any of six DNA mismatch repair genes (ATM, ERCC2, FANCD2, PALB2, BRCA1 or BRCA2) not only had a high somatic mutation burden, but also had enhanced recurrence-free survival compared to non-carriers of these mutations. We hypothesize that a high somatic mutation burden may generate a higher number of tumor-specific antigens that induce clonal expansion of tumor-infiltrating T-lymphocytes (TILs) to reduce a risk of recurrence and improve the survival. To investigate this hypothesis, we have sequenced the TCR cDNAs of TILs.

Methods: Tumor and adjacent normal tissue (verified by a pathologist) were collected from patients whose tumors had previously been whole-exome sequenced. Library preparation was performed using previously described methods. Next-generation sequencing of TCRs was conducted on the Illumina platform (San Diego, CA) and CDR3 sequences were identified using a published algorithm. The diversity index of the TCRα and TCRβ chains was calculated using Simpson's diversity index, an ecological formula for calculating biodiversity based on evenness and richness. CD4+, CD8+, and FOXP3 gene expression levels were also examined with TaqMan Gene Expression Assays (Thermo Fisher Scientific, Carlsbad, CA).

Results: TCR sequencing of ten tumors (5 non-recurrent (NR) with at least two years follow-up, 5 recurrent) has been performed. The average TCRα DI of five recurrent patients is 97±56.5 (SEM) compared to 23± 12.7 for five NR patients. In addition, we found an average DI of 13.8±0.77 in tumors with a high somatic mutation burden (average number of mutations 221), while it was 71.4±36.8 for tumors with a low somatic mutation burden (avg mutations 31). In samples with a relatively high DI, the three most abundant TCR clones represent less than 15% of the total TCR population, while in samples with a low DI, the top three clones comprise 35-90% of the total population. Finally, the CD8+/FOXP3 ratios were 10.1±2.1(SEM) and 5.6±2.9 for tumors with a low DI and those with a high DI, respectively (low vs. high defined by the average).

Conclusions: We have elicited a correlating pattern where MIBC tumors with a higher number of somatic mutations have a lower TCR DI, indicating an oligoclonal expansion. Furthermore, in patients with recurrence, the TCR DI is higher, suggesting a lack of anti-tumor immune responses and non-specific T-cell expansion. The higher CD8+/FOXP3 ratio observed in tumors with low DI suggests that the clonal expansion is of CD8+ cells in tumors, which have been shown to be associated with favorable prognosis.

Citation Format: Noura Choudhury, Kai Lee Yap, Kazuma Kiyotani, Poh Yin Yew, Alexa Campanile, Tatjana Antic, Gary Steinberg, Jae-Hyun Park, Peter H. O'Donnell, Yusuke Nakamura. Tumor T-cell receptor (TCR) diversity elucidates the immune response to genetic alterations of muscle-invasive bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4899. doi:10.1158/1538-7445.AM2015-4899