Single-Cell Quantification of mRNA Expression in The Human Brain

RNA analysis at the cellular resolution in the human brain is challenging. Here, we describe an optimised approach for detecting single RNA transcripts in a cell-type specific manner in frozen human brain tissue using multiplexed fluorescent RNAscope probes. We developed a new robust analytical approach for RNAscope quantification. Our method shows that low RNA integrity does not significantly affect RNAscope signal, recapitulates bulk RNA analysis and provides spatial context to transcriptomic analysis of human post-mortem brain at single-cell resolution. In summary, our optimised method allows the usage of frozen human samples from brain banks to perform quantitative RNAscope analysis

A transcriptionally and functionally distinct PD-1⁺ CD8⁺ T cell pool with predictive potential in non-small-cell lung cancer treated with PD-1 blockade.

Evidence from mouse chronic viral infection models suggests that CD8 <sup>+</sup> T cell subsets characterized by distinct expression levels of the receptor PD-1 diverge in their state of exhaustion and potential for reinvigoration by PD-1 blockade. However, it remains unknown whether T cells in human cancer adopt a similar spectrum of exhausted states based on PD-1 expression levels. We compared transcriptional, metabolic and functional signatures of intratumoral CD8 <sup>+</sup> T lymphocyte populations with high (PD-1 <sup>T</sup> ), intermediate (PD-1 <sup>N</sup> ) and no PD-1 expression (PD-1 <sup>-</sup> ) from non-small-cell lung cancer patients. PD-1 <sup>T</sup> T cells showed a markedly different transcriptional and metabolic profile from PD-1 <sup>N</sup> and PD-1 <sup>-</sup> lymphocytes, as well as an intrinsically high capacity for tumor recognition. Furthermore, while PD-1 <sup>T</sup> lymphocytes were impaired in classical effector cytokine production, they produced CXCL13, which mediates immune cell recruitment to tertiary lymphoid structures. Strikingly, the presence of PD-1 <sup>T</sup> cells was strongly predictive for both response and survival in a small cohort of non-small-cell lung cancer patients treated with PD-1 blockade. The characterization of a distinct state of tumor-reactive, PD-1-bright lymphocytes in human cancer, which only partially resembles that seen in chronic infection, provides potential avenues for therapeutic intervention

Tumour invasiveness, the local and systemic environment and the basis of staging systems in colorectal cancer

background: The present study aimed to examine the relationship between tumour invasiveness (T stage), the local and systemic environment and cancer-specific survival (CSS) of patients with primary operable colorectal cancer. methods: The tumour microenvironment was examined using measures of the inflammatory infiltrate (Klintrup-Makinen (KM) grade and Immunoscore), tumour stroma percentage (TSP) and tumour budding. The systemic inflammatory environment was examined using modified Glasgow Prognostic Score (mGPS) and neutrophil:lymphocyte ratio (NLR). A 5-year CSS was examined. results: A total of 331 patients were included. Increasing T stage was associated with colonic primary, N stage, poor differentiation, margin involvement and venous invasion (P<0.05). T stage was significantly associated with KM grade (P=0.001), Immunoscore (P=0.016), TSP (P=0.006), tumour budding (P<0.001), and elevated mGPS and NLR (both P<0.05). In patients with T3 cancer, N stage stratified survival from 88 to 64%, whereas Immunoscore and budding stratified survival from 100 to 70% and from 91 to 56%, respectively. The Glasgow Microenvironment Score, a score based on KM grade and TSP, stratified survival from 93 to 58%. conclusions: Although associated with increasing T stage, local and systemic tumour environment characteristics, and in particular Immunoscore, budding, TSP and mGPS, are stage-independent determinants of survival and may be utilised in the staging of patients with primary operable colorectal cancer

PD-1T TILs as a predictive biomarker for clinical benefit to PD-1 blockade in patients with advanced NSCLC

PURPOSE Durable clinical benefit to PD-1 blockade in NSCLC is currently limited to a small fraction of patients, underlining the need for predictive biomarkers. We recently identified a tumor-reactive tumor-infiltrating T lymphocyte (TIL) pool, termed PD-1T TILs, with predictive potential in NSCLC. Here, we examined PD-1T TILs as biomarker in NSCLC. EXPERIMENTAL DESIGN PD-1T TILs were digitally quantified in120 baseline samples from advanced NSCLC patients treated with PD-1 blockade. Primary outcome was Disease Control (DC) at 6 months. Secondary outcomes were DC at 12 months and survival. Exploratory analyses addressed the impact of lesion-specific responses, tissue sample properties and combination with other biomarkers on the predictive value of PD-1T TILs. RESULTS PD-1T TILs as a biomarker reached 77% sensitivity and 67% specificity at 6 months, and 93% and 65% at 12 months, respectively. Particularly, a patient group without clinical benefit was reliably identified, indicated by a high negative predictive value (NPV) (88% at 6 months, 98% at 12 months). High PD-1T TILs related to significantly longer progression-free (HR 0.39, 95% CI: 0.24-0.63, p<0.0001) and overall survival (HR 0.46, 95% CI: 0.28-0.76, p<0.01). Predictive performance was increased when lesion-specific responses and samples obtained immediately before treatment were assessed. Notably, the predictive performance of PD-1TTILs was superior to PD-L1 and TLS in the same cohort. CONCLUSIONS This study established PD-1T TILs as predictive biomarker for clinical benefit to PD-1 blockade in advanced NSCLC patients. Most importantly, the high NPV demonstrates an accurate identification of a patient group without benefit

Prognostic image-based quantification of CD8CD103 T cell subsets in high-grade serous ovarian cancer patients

CD103-positive tissue resident memory-like CD8+ T cells (CD8CD103 TRM) are associated with improved prognosis across malignancies, including high-grade serous ovarian cancer (HGSOC). However, whether quantification of CD8, CD103 or both is required to improve existing survival prediction and whether all HGSOC patients or only specific subgroups of patients benefit from infiltration, remains unclear. To address this question, we applied image-based quantification of CD8 and CD103 multiplex immunohistochemistry in the intratumoral and stromal compartments of 268 advanced-stage HGSOC patients from two independent clinical institutions. Infiltration of CD8CD103 immune cell subsets was independent of clinicopathological factors. Our results suggest CD8CD103 TRM quantification as a superior method for prognostication compared to single CD8 or CD103 quantification. A survival benefit of CD8CD103 TRM was observed only in patients treated with primary cytoreductive surgery. Moreover, survival benefit in this group was limited to patients with no macroscopic tumor lesions after surgery. This approach provides novel insights into prognostic stratification of HGSOC patients and may contribute to personalized treatment strategies in the future

Critical research gaps and recommendations to inform research prioritisation for more effective prevention and improved outcomes in colorectal cancer

OBJECTIVE: Colorectal cancer (CRC) leads to significant morbidity/mortality worldwide. Defining critical research gaps (RG), their prioritisation and resolution, could improve patient outcomes.DESIGN: RG analysis was conducted by a multidisciplinary panel of patients, clinicians and researchers (n=71). Eight working groups (WG) were constituted: discovery science; risk; prevention; early diagnosis and screening; pathology; curative treatment; stage IV disease; and living with and beyond CRC. A series of discussions led to development of draft papers by each WG, which were evaluated by a 20-strong patient panel. A final list of RGs and research recommendations (RR) was endorsed by all participants.RESULTS: Fifteen critical RGs are summarised below: RG1: Lack of realistic models that recapitulate tumour/tumour micro/macroenvironment; RG2: Insufficient evidence on precise contributions of genetic/environmental/lifestyle factors to CRC risk; RG3: Pressing need for prevention trials; RG4: Lack of integration of different prevention approaches; RG5: Lack of optimal strategies for CRC screening; RG6: Lack of effective triage systems for invasive investigations; RG7: Imprecise pathological assessment of CRC; RG8: Lack of qualified personnel in genomics, data sciences and digital pathology; RG9: Inadequate assessment/communication of risk, benefit and uncertainty of treatment choices; RG10: Need for novel technologies/interventions to improve curative outcomes; RG11: Lack of approaches that recognise molecular interplay between metastasising tumours and their microenvironment; RG12: Lack of reliable biomarkers to guide stage IV treatment; RG13: Need to increase understanding of health related quality of life (HRQOL) and promote residual symptom resolution; RG14: Lack of coordination of CRC research/funding; RG15: Lack of effective communication between relevant stakeholders.CONCLUSION: Prioritising research activity and funding could have a significant impact on reducing CRC disease burden over the next 5 years.</p

In-depth clinical and biological exploration of DNA Damage Immune Response (DDIR) as a biomarker for oxaliplatin use in colorectal cancer

PURPOSE: The DNA Damage Immune Response (DDIR) assay was developed in breast cancer (BC) based on biology associated with deficiencies in homologous recombination and Fanconi Anemia (HR/FA) pathways. A positive DDIR call identifies patients likely to respond to platinum-based chemotherapies in breast and oesophageal cancers. In colorectal cancer (CRC) there is currently no biomarker to predict response to oxaliplatin. We tested the ability of the DDIR assay to predict response to oxaliplatin-based chemotherapy in CRC and characterised the biology in DDIR-positive CRC. METHODS: Samples and clinical data were assessed according to DDIR status from patients who received either 5FU or FOLFOX within the FOCUS trial (n=361, stage 4), or neo-adjuvant FOLFOX in the FOxTROT trial (n=97, stage 2/3). Whole transcriptome, mutation and immunohistochemistry data of these samples were used to interrogate the biology of DDIR in CRC. RESULTS: Contrary to our hypothesis, DDIR negative patients displayed a trend towards improved outcome for oxaliplatin-based chemotherapy compared to DDIR positive patients. DDIR positivity was associated with Microsatellite Instability (MSI) and Colorectal Molecular Subtype 1 (CMS1). Refinement of the DDIR signature, based on overlapping interferon-related chemokine signalling associated with DDIR positivity across CRC and BC cohorts, further confirmed that the DDIR assay did not have predictive value for oxaliplatin-based chemotherapy in CRC. CONCLUSIONS: DDIR positivity does not predict improved response following oxaliplatin treatment in CRC. However, data presented here suggests the potential of the DDIR assay in identifying immune-rich tumours that may benefit from immune checkpoint blockade, beyond current use of MSI status

Tumor-associated macrophages and response to 5-flourouracil adjuvant therapy in stage III colorectal cancer

Tumor-associated macrophages (TAMs) play a role in tumor development and progression. We hypothesized that abundance of TAMs might modify efficacy of 5-fluorouracil chemotherapy in colorectal cancer. We measured the density of CD68+ TAMs at the invasive front of primary tumor of colorectal carcinoma (PT-TAMs; n = 208), at available matched metastatic lymph node (LN-TAMs; n = 149), and in an independent set of primary colorectal cancers (PT-TAMs, n = 111). The hazard ratios for disease-free survival were computed by Cox proportional-hazards model. In exploratory analysis, the interaction between TAMs and 5-fluorouracil adjuvant therapy was significant (PT-TAMs, p = 0.02; LN-TAMs, p = 0.005). High TAMs were independently associated with better disease-free survival only in 5-fluorouracil-treated patients (PT-TAMs, HR 0.23; 95%CI, 0.08\ue2\u80\u930.65; p = 0.005; LN-TAMs, HR 0.13; 95%CI, 0.04\ue2\u80\u930.43; p = 0.001). The independent predictive value of PT-TAMs was replicated in the external set (HR, 0.14; 95%CI 0.02\ue2\u80\u931.00; p = 0.05). In an in vitro experiment, 5-fluorouracil and macrophages showed a synergistic effect and increased colorectal cancer cell death. High densities of TAMs, particularly in metastatic lymph-nodes, identify stage III colorectal cancer patients benefitting from 5-fluorouracil adjuvant therapy