Serial sampling of rectal tumors during radiotherapy: A proof-of-concept study.
Authors: Jones Leia , Pennel Kathryn AF , Quinn Jean A. , Magill Abbey , Nair Harikrishnan S. , McMahon Ross K , McDonald Alec , Graham Janet , Wood Colin , Amrikhah Raheleh , Horgan Paul G. , Steele Colin William , Dunne Philip D , Edwards Joanne , O'Cathail Sean Michael , Roxburgh Campbell SD 
Published: January 2022

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Background: Treatment response to neoadjuvant therapy in locally advanced rectal cancer (LARC) remains heterogenous. Clinicians are guided by pre-treatment clinical assessment alone in determining neoadjuvant strategy. More must be done to uncover biological mechanisms underpinning response and resistance. We have developed a biospecimen collection protocol in LARC performing serial sampling of tumors and peripheral blood samples prior to, during and after treatment to characterize the biological evolution of this heterogenous response. Here we present early proof of concept results with a focus on the intra-tumoral immune response relating to radiotherapy (RT). Methods: Patients receiving standard-of-care neoadjuvant RT were recruited to an ethically approved study between Dec 2018 - Aug 2021. The protocol consisted of a baseline biopsy and blood sample prior to RT followed by repeat sampling at 2, 6 and 12wks after Day 1 of RT. Standard immunohistochemistry (IHC) was performed for markers of immune activity. Target capture sequencing was performed using RNA baits extracted from serial biopsies to target a 276 genes panel. Paired tumor-normal sequencing was performed. Bulk 3’ RNA seq (Lexogen Illumina Quantseq) characterized immune and inflammatory gene expression. A multiplex bead array (Luminex xPONENT) of 24 cytokines and chemokines was performed using serial plasma samples. Results: 17pts were recruited, 3 with stage IV disease. 14pts received chemoradiation and 3 pts had short-course based regimens. Treatment responses were evaluable in 14pts: graded complete in 2pts; good/ near complete in 7pts and partial/ poor in 5pts. All tumors were MSS, and most frequently mutated genes were APC (75%), KRAS (38%), NRAS (25%) , NOTCH1 (25%) and PIK3CA (25%) (n = 8). Quantseq demonstrated that the immune/ inflammatory response, as measured by interferon-gamma response and IL-6/ JAK-STAT signaling, was significantly elevated up to 12wks after Day 1 RT, with a peak at around 6wks (n = 3). Correlative IHC showed an increase in innate immune cells in pts with a favorable response at 6wks (n = 8). Cytokine/ chemokine analysis suggested patients with a favorable response demonstrated strong inflammatory (MCP1 & IL-17a) responses 2 and 6wks post-RT and strong CTL (Granzyme B) and Th1 (GM-CSF & IP-10) responses 12wks post-RT (n = 10). Conclusions: We show acquisition of meaningful genomic and transcriptomic material from serial biopsies in rectal cancer is possible. Early data suggest that dynamic profiling of rectal tumors demonstrates transcriptomic evolution during treatment. Specifically, we show that the immune response to radiotherapy peaks at around 6wks after initiation of RT and persists to 12wks. This supports ongoing trials of immunomodulatory treatments in combination with, and following, RT in rectal cancer. Further work is required to define differences between responders and non-responders.