Insights into the metastatic cascade through research autopsies

Metastasis is a complex process and the leading cause of cancer-related death globally. Recent studies have demonstrated that genomic sequencing data from paired primary and metastatic tumours can be used to trace the evolutionary origins of cells responsible for metastasis. This approach has yielded new insights into the genomic alterations that engender metastatic potential, and the mechanisms by which cancer spreads. Given that the reliability of these approaches is contingent upon how representative the samples are of primary and metastatic tumour heterogeneity, we review insights from studies that have reconstructed the evolution of metastasis within the context of their cohorts and designs. We discuss the role of research autopsies in achieving the comprehensive sampling necessary to advance the current understanding of metastasis

Trans-arterial chemoembolization as a loco-regional inducer of immunogenic cell death in hepatocellular carcinoma: implications for immunotherapy

Immunotherapy; Liver neoplasmsInmunoterapia; Neoplasias hepáticasImmunoteràpia; Neoplàsies hepàtiquesBackground Modulation of adaptive immunity may underscore the efficacy of trans-arterial chemoembolization (TACE). We evaluated the influence of TACE on T-cell function by phenotypic lymphocyte characterization in samples of patients undergoing surgery with (T+) or without (T-) prior-TACE treatment. Methods We profiled intratumoral (IT), peritumoral (PT) and non-tumoral (NT) background tissue to evaluate regulatory CD4+/FOXP3+ (T-reg) and immune-exhausted CD8+/PD-1+ T-cells across T+ (n=58) and T− (n=61). We performed targeted transcriptomics and T-cell receptor sequencing in a restricted subset of samples (n=24) evaluated in relationship with the expression of actionable drivers of anti-cancer immunity including PD-L1, indoleamine 2,3 dehydrogenase (IDO-1), cytotoxic T-lymphocyte associated protein 4 (CTLA-4), Lag-3, Tim-3 and CD163. Results We analyzed 119 patients resected (n=25, 21%) or transplanted (n=94, 79%) for Child-Pugh A (n=65, 55%) and Barcelona Clinic Liver Cancer stage A (n=92, 77%) hepatocellular carcinoma. T+ samples displayed lower IT CD4+/FOXP3+ (p=0.006), CD8+ (p=0.002) and CD8+/PD-1+ and NT CD8+/PD-1+ (p<0.001) compared with T−. Lower IT (p=0.005) and NT CD4+/FOXP3+ (p=0.03) predicted for improved recurrence-free survival. In a subset of samples (n=24), transcriptomic analysis revealed upregulation of a pro-inflammatory response in T+. T+ samples were enriched for IRF2 expression (p=0.01), an interferon-regulated transcription factor implicated in cancer immune-evasion. T-cell clonality and expression of PD-L1, IDO-1, CTLA-4, Lag-3, Tim-3 and CD163 was similar in T+ versus T−. Conclusions TACE is associated with lower IT density of immune-exhausted effector cytotoxic and T-regs, with significant upregulation of pro-inflammatory pathways. This highlights the pleiotropic effects of TACE in modulating the tumor microenvironment and strengthens the rationale for developing immunotherapy alongside TACE.DJP is supported by grant funding from the Wellcome Trust Strategic Fund (PS3416) and by the Cancer Research UK Postdoctoral bursary (C57701/A26137). AF is supported by grant from Instituto de Salud Carlos III (PI18/00542)

T-cell mediated responses against alpha-foetoprotein in hepatocellular carcinoma: Relationship with hepatitis C virus infection, tumour phenotype and patients’ survival

Background Alpha-foetoprotein (AFP) is a potential immunotherapeutic target in hepatocellular carcinoma (HCC). However, T-cell response (TR) to AFP is suppressed in HCC due to immune evasion. It is unknown whether HCV infection may pre-condition TR against AFP, or whether TR may influence the clinical course of HCC. Methods We prospectively enrolled 18 HCV+ treatment-naïve patients with cirrhosis (CC), 18 HCV+ HCC cases and 17 HCV- HCC cases. TR was quantified by ELISPOT using assays specific to interleukin (IL) 2, IL10 and granulocyte-monocyte colony stimulating factor (GM-CSF) on ex-vivo peripheral blood mononuclear cells (PBMC) stimulated in vitro with AFP peptides. Cytokine ratios were compared between groups and with clinicopathological features of HCC, including overall survival (OS). Results The proportion of AFP-specific responses was not different across the studied groups for any of the assayed cytokines. AFP-specific IL-2 responses were increased in larger (P = .02), multifocal tumours (P = .01) and correlated with advanced disease (P = .01). TRs did not correlate with other clinicopathological factors and did not predict for OS. Conclusion Tumour stage but not HCV infection is related to the emergence of anti-AFP TRs. These data enable formulation of a rationale for the further development of anti-AFP immunotherapy in HCC, facilitating optimal patient selection for future studies

Risk stratification of Barrett's oesophagus using a non-endoscopic sampling method coupled with a biomarker panel: a cohort study.

BACKGROUND: Barrett's oesophagus predisposes to adenocarcinoma. However, most patients with Barrett's oesophagus will not progress and endoscopic surveillance is invasive, expensive, and fraught by issues of sampling bias and the subjective assessment of dysplasia. We investigated whether a non-endoscopic device, the Cytosponge, could be coupled with clinical and molecular biomarkers to identify a group of patients with low risk of progression suitable for non-endoscopic follow-up. METHODS: In this multicentre cohort study (BEST2), patients with Barrett's oesophagus underwent the Cytosponge test before their surveillance endoscopy. We collected clinical and demographic data and tested Cytosponge samples for a molecular biomarker panel including three protein biomarkers (P53, c-Myc, and Aurora kinase A), two methylation markers (MYOD1 and RUNX3), glandular atypia, and TP53 mutation status. We used a multivariable logistic regression model to compute the conditional probability of dysplasia status. We selected a simple model with high classification accuracy and applied it to an independent validation cohort. The BEST2 study is registered with ISRCTN, number 12730505. FINDINGS: The discovery cohort consisted of 468 patients with Barrett's oesophagus and intestinal metaplasia. Of these, 376 had no dysplasia and 22 had high-grade dysplasia or intramucosal adenocarcinoma. In the discovery cohort, a model with high classification accuracy consisted of glandular atypia, P53 abnormality, and Aurora kinase A positivity, and the interaction of age, waist-to-hip ratio, and length of the Barrett's oesophagus segment. 162 (35%) of 468 of patients fell into the low-risk category and the probability of being a true non-dysplastic patient was 100% (99% CI 96-100) and the probability of having high-grade dysplasia or intramucosal adenocarcinoma was 0% (0-4). 238 (51%) of participants were classified as of moderate risk; the probability of having high-grade dysplasia was 14% (9-21). 58 (12%) of participants were classified as high-risk; the probability of having non-dysplastic endoscopic biopsies was 13% (5-27), whereas the probability of having high-grade dysplasia or intramucosal adenocarcinoma was 87% (73-95). In the validation cohort (65 patients), 51 were non-dysplastic and 14 had high-grade dysplasia. In this cohort, 25 (38%) of 65 patients were classified as being low-risk, and the probability of being non-dysplastic was 96·0% (99% CI 73·80-99·99). The moderate-risk group comprised 27 non-dysplastic and eight high-grade dysplasia cases, whereas the high-risk group (8% of the cohort) had no non-dysplastic cases and five patients with high-grade dysplasia. INTERPRETATION: A combination of biomarker assays from a single Cytosponge sample can be used to determine a group of patients at low risk of progression, for whom endoscopy could be avoided. This strategy could help to avoid overdiagnosis and overtreatment in patients with Barrett's oesophagus. FUNDING: Cancer Research UK.The BEST2 study was funded by Cancer Research UKThis is the author accepted manuscript. The final version is available from Elsevier via https://doi.org/10.1016/S2468-1253(16)30118-

Early Antibiotic Exposure Is Not Detrimental to Therapeutic Effect from Immunotherapy in Hepatocellular Carcinoma

BACKGROUND AND RATIONALE: Immune checkpoint inhibitor (ICI) therapy is an expanding therapeutic option for hepatocellular carcinoma (HCC). Antibiotics (ATB) taken prior to or early during ICI therapy can impact immunotherapy efficacy across indications; however, the effect of ATB is undefined in HCC. METHODS: In a large international cohort of 450 ICI recipients from Europe, North America, and Asia, we categorized patients according to timing of ATB focusing on exposure within −30 to +30 days from ICI (early immunotherapy period [EIOP]). EIOP was evaluated in association with overall survival (OS), progression-free survival (PFS), and best radiologic response using RECIST 1.1 criteria. RESULTS: Our study comprised mostly cirrhotic (329, 73.3%) males (355, 79.1%) with a Child-Turcotte Pugh class of A (332, 73.9%), receiving ICI after 1 therapy line (251, 55.9%) for HCC of Barcelona clinic liver cancer stage C (325, 72.4%). EIOP (n = 170, 37.9%) was independent of baseline clinicopathologic features of HCC and correlated with longer PFS (6.1 vs. 3.7 months, log-rank p = 0.0135). EIOP+ patients had similar OS, overall response, and disease control rates (DCRs) compared to EIOP. The effect of EIOP persisted in landmark time analyses and in multivariable models, confirming the independent predictive role of EIOP in influencing PFS following adjustment for covariates reflective of tumor burden, liver function, and ICI regimen administered. In patients receiving programmed cell death-1 receptor/ligand inhibitors monotherapy, EIOP was also associated with higher DCRs (61.4% vs. 50.9%, p = 0.0494). CONCLUSIONS: Unlike other oncological indications, ATB in the 30 days before or after ICI initiation is associated with improved benefit from immunotherapy, independent of disease and treatment-related features. Evaluation of the immune microbiologic determinants of response to ICI in HCC warrants further investigation

Phenotypic Characteristics of the Tumour Microenvironment in Primary and Secondary Hepatocellular Carcinoma

(1) Background: The intra-tumoural heterogeneity (ITH) of hepatocellular carcinoma (HCC) and its microenvironment (TME) across primary and secondary disease is poorly characterised. (2) Methods: Intra-tumoural (IT) and peri-tumoural (PT) staining of matched primary and secondary samples was conducted to evaluate the distribution of CD4+/FOXP3+ and CD8+/PD1+ T-cells. Samples underwent PD-L1/2 immunostaining, tumour mutational burden (TMB) evaluation, and high-resolution T-cell receptor (TCR) sequencing to derive T-cell clonality and targeted transcriptomics. (3) Results: We analysed 24 samples from matched primary (n = 11) and secondary (n = 13; 5 synchronous, 6 metachronous) deposits, 11 being extrahepatic (84.6%). IT CD8+ density was lower than PT in both primary (p = 0.005) and secondary deposits (p = 0.01), consistent with immune exclusion. PD-L1+ tumours displayed higher IT and PT CD8+/PD1+ cell density compared to PD-L1- (p &lt; 0.05), and primary IT infiltrate was enriched in CD4+/FOXP3+ cells, compared to PT regions (p = 0.004). TCR-sequencing demonstrated enrichment of the top T-cell clonotype in secondary versus primary HCC (p = 0.02), without differences in overall productive clonality (p = 0.35). TMB was similar across primary versus secondary HCC (p = 0.95). While directed gene set analysis demonstrated the uniformity of transcriptional signatures of individual immune cell types, secondary deposits demonstrated higher COLEC12 (p = 0.004), CCL26 (p = 0.02), CD1E (p = 0.02) and CD36 (p = 0.03) expression with downregulation of CXCL1 (p = 0.03), suggesting differential regulation of innate immunity. (4) Conclusion: Immune exclusion is a defining feature of the HCC TME. Despite evidence of homogeneity in somatic TMB, secondary HCC is characterised by the expansion of a distinct T-cell clonotype and differential regulation of innate immune pathways

Evaluation of the sensitivity of R1ρ MRI to pH and macromolecular density.

The tumor microenvironment is characteristically acidic and this extracellular acidosis is known to play a role in carcinogenesis and metastasis and can affect tumor chemosensitivity and radiosensitivity. Intracellular pH has been used as a possible biomarker of salvageable tissue in ischemic stroke. A non-invasive MRI-based approach for the determination and imaging of cerebral pH would be a powerful tool in cancer diagnosis and monitoring, as well as stroke treatment planning. Several pH-based MRI imaging approaches have been proposed but for these to be useful, disentangling the effects of pH from other parameters which may affect the measured MRI signal is crucial to ensure accuracy and specificity. R1 relaxation in the rotating frame (R1ρ) is an example of a method that has been proposed to probe pH in vivo using MRI. In this study, we have investigated the relationship between R1ρ, pH, and macromolecular density in vitro using phantoms and in human volunteers. Here we show that the rate of R1ρ relaxation (=1/T1ρ) varies with pH but only in the presence of macromolecules. At constant pH, phantom macromolecular density inversely correlated with R1ρ. R1ρ imaging of the normal human brain demonstrated regional heterogeneity with significant differences between structurally distinct regions, which are likely to be independent of pH. For example, R1ρ was higher in the basal ganglia compared to grey matter and higher in grey matter compared to white matter. We conclude that R1ρ cannot be reliably used to image tissue pH without deconvolution from the effects of local tissue macromolecular composition