Cytotoxic T-cell exhaustion analyses by in situ single-cell immunofluorescence in relation to colorectal cancer patient age, tissue bacteria and mortality
Nobuhiro Nakazawa, Kosuke Matsuda, Satoko Ugai, Satoshi Miyahara, Atsushi Kondo, Mayu Higashioka, Yuxue Zhong, Alessandro Mannucci, Giulia Martina Cavestro, Andrew T Chan, Jonathan A Nowak, Mingyang Song, Hiroshi Saeki, Marios Giannakis, Juha P Väyrynen, Tomotaka Ugai, Shuji OginoObjectives
We hypothesised that cytotoxic T-cell exhaustion status in colorectal cancer (CRC) microenvironment relates to age of diagnosis (eg, early-onset CRC), pathogenic bacteria and clinical outcomes.
Design
Population-based prospective cohort study using the prospective cohort incident-tumour biobank method (PCIBM).
Setting
Two US nationwide prospective cohort studies, the Nurses’ Health Study (NHS) and the Health Professionals Follow-Up Study (HPFS).
Participants
Among 4476 incident CRC cases identified during follow-up, 857 patients with available tumour tissue were included in the present tissue-based analysis. Patients with colon and rectal adenocarcinomas were included.
Interventions
Not applicable.
Main outcome measures
In situ single-cell multispectral immunofluorescence combined with computational machine learning for CD3, CD8, CD274 (PD-L1), HAVCR2 (TIM-3), PDCD1 (PD-1) and KRT (keratin) was used to characterise non-exhausted, progenitor-exhausted and exhausted CD3 + CD8 + cells according to PDCD1 (PD-1) and HAVCR2 (TIM-3) expression. Primary measures included densities of CD3 + CD8 + cell exhaustion subsets and their associations with clinicopathological characteristics, age at CRC diagnosis, selected tumour tissue bacteria, CRC-specific mortality and spatial proximity to tumour cells.
Results
Younger age of CRC diagnosis generally correlated with lower densities of CD3 + CD8 + cells. Multivariable-adjusted ORs (with 95% CI) in age <55 (vs age ≥70) for overall, progenitor-exhausted and exhausted CD3 + CD8 + cell stromal densities (all quartiles) were 0.39 (0.22 to 0.69; P trend =0.0033), 0.40 (0.23 to 0.68; P trend =0.0048) and 0.44 (0.25 to 0.78; P trend =0.0082), respectively. Multivariable-adjusted ORs (with 95% CI) for stromal non-exhausted CD3 + CD8 + cells (quartiles) were 0.81 (0.56 to 1.17) and 0.52 (0.37 to 0.74) (P trend =0.0004) for low-level and high-level Bacteroides fragilis (vs negativity), respectively. Multivariable-adjusted ORs for stromal exhausted CD3 + CD8 + cells (quartiles) were 0.37 (0.19 to 0.72) and 0.60 (0.30 to 1.17) (P trend =0.0081) for low-level and high-level enterotoxigenic Bacteroides fragilis (vs negativity), respectively. Multivariable-adjusted CRC-specific mortality HRs (with 95% CI) in quartile 4 (Q4; vs Q1) of progenitor-exhausted CD3 + CD8 + cells in the intraepithelial region and their proximity to tumour cells were 0.42 (0.26 to 0.67; P trend <0.0001) and 0.50 (0.32 to 0.78; P trend =0.0004), respectively.
Conclusion
This PCIBM-based study provides evidence that progenitor-exhausted CD3 + CD8 + cell density and their proximity to tumour cells are favourable prognostic biomarkers in CRC. Moreover, lower stromal densities of overall, progenitor-exhausted and exhausted CD3 + CD8 + cells are associated with younger-onset CRC.