Multiplex analysis of intratumoural immune infiltrate and prognosis in patients with stage II-III colorectal cancer from the SCOT and QUASAR 2 trials: a retrospective analysis.

BACKGROUND: Tumour-infiltrating CD8+ cytotoxic T cells confer favourable prognosis in colorectal cancer. The added prognostic value of other infiltrating immune cells is unclear and so we sought to investigate their prognostic value in two large clinical trial cohorts. METHODS: We used multiplex immunofluorescent staining of tissue microarrays to assess the densities of CD8+, CD20+, FoxP3+, and CD68+ cells in the intraepithelial and intrastromal compartments from tumour samples of patients with stage II-III colorectal cancer from the SCOT trial (ISRCTN59757862), which examined 3 months versus 6 months of adjuvant oxaliplatin-based chemotherapy, and from the QUASAR 2 trial (ISRCTN45133151), which compared adjuvant capecitabine with or without bevacizumab. Both trials included patients aged 18 years or older with an Eastern Cooperative Oncology Group performance status of 0-1. Immune marker predictors were analysed by multiple regression, and the prognostic and predictive values of markers for colorectal cancer recurrence-free interval by Cox regression were assessed using the SCOT cohort for discovery and QUASAR 2 cohort for validation. FINDINGS: After exclusion of cases without tissue microarrays and with technical failures, and following quality control, we included 2340 cases from the SCOT trial and 1069 from the QUASAR 2 trial in our analysis. Univariable analysis of associations with recurrence-free interval in cases from the SCOT trial showed a strong prognostic value of intraepithelial CD8 (CD8IE) as a continuous variable (hazard ratio [HR] for 75th vs 25th percentile [75vs25] 0·73 [95% CI 0·68-0·79], p=2·5 × 10-16), and of intrastromal FoxP3 (FoxP3IS; 0·71 [0·64-0·78], p=1·5 × 10-13) but not as strongly in the epithelium (FoxP3IE; 0·89 [0·84-0·96], p=1·5 × 10-4). Associations of other markers with recurrence-free interval were moderate. CD8IE and FoxP3IS retained independent prognostic value in bivariable and multivariable analysis, and, compared with either marker alone, a composite marker including both markers (CD8IE-FoxP3IS) was superior when assessed as a continuous variable (adjusted [a]HR75 vs 25 0·70 [95% CI 0·63-0·78], p=5·1 × 10-11) and when categorised into low, intermediate, and high density groups using previously published cutpoints (aHR for intermediate vs high 1·68 [95% CI 1·29-2·20], p=1·3 × 10-4; low vs high 2·58 [1·91-3·49], p=7·9 × 10-10), with performance similar to the gold-standard Immunoscore. The prognostic value of CD8IE-FoxP3IS was confirmed in cases from the QUASAR 2 trial, both as a continuous variable (aHR75 vs 25 0·84 [95% CI 0·73-0·96], p=0·012) and as a categorical variable for low versus high density (aHR 1·80 [95% CI 1·17-2·75], p=0·0071) but not for intermediate versus high (1·30 [0·89-1·88], p=0·17). INTERPRETATION: Combined evaluation of CD8IE and FoxP3IS could help to refine risk stratification in colorectal cancer. Investigation of FoxP3IS cells as an immunotherapy target in colorectal cancer might be merited. FUNDING: Medical Research Council, National Institute for Health Research, Cancer Research UK, Swedish Cancer Society, Roche, and Promedica Foundation.

PRMT5 inhibition shows in vitro efficacy against H3K27M-altered diffuse midline glioma, but does not extend survival in vivo.

H3K27-altered Diffuse Midline Glioma (DMG) is a universally fatal paediatric brainstem tumour. The prevalent driver mutation H3K27M creates a unique epigenetic landscape that may also establish therapeutic vulnerabilities to epigenetic inhibitors. However, while HDAC, EZH2 and BET inhibitors have proven somewhat effective in pre-clinical models, none have translated into clinical benefit due to either poor blood-brain barrier penetration, lack of efficacy or toxicity. Thus, there remains an urgent need for new DMG treatments. Here, we performed wider screening of an epigenetic inhibitor library and identified inhibitors of protein arginine methyltransferases (PRMTs) among the top hits reducing DMG cell viability. Two of the most effective inhibitors, LLY-283 and GSK591, were targeted against PRMT5 using distinct binding mechanisms and reduced the viability of a subset of DMG cells expressing wild-type TP53 and mutant ACVR1. RNA-sequencing and phenotypic analyses revealed that LLY-283 could reduce the viability, clonogenicity and invasion of DMG cells in vitro, representing three clinically important phenotypes, but failed to prolong survival in an orthotopic xenograft model. Together, these data show the challenges of DMG treatment and highlight PRMT5 inhibitors for consideration in future studies of combination treatments.

Cervical lymph nodes and ovarian teratomas as germinal centres in NMDA receptor-antibody encephalitis.

Autoantibodies against the extracellular domain of the N-methyl-d-aspartate receptor (NMDAR) NR1 subunit cause a severe and common form of encephalitis. To better understand their generation, we aimed to characterize and identify human germinal centres actively participating in NMDAR-specific autoimmunization by sampling patient blood, CSF, ovarian teratoma tissue and, directly from the putative site of human CNS lymphatic drainage, cervical lymph nodes. From serum, both NR1-IgA and NR1-IgM were detected more frequently in NMDAR-antibody encephalitis patients versus controls (both P < 0.0001). Within patients, ovarian teratoma status was associated with a higher frequency of NR1-IgA positivity in serum (OR = 3.1; P < 0.0001) and CSF (OR = 3.8, P = 0.047), particularly early in disease and before ovarian teratoma resection. Consistent with this immunoglobulin class bias, ovarian teratoma samples showed intratumoral production of both NR1-IgG and NR1-IgA and, by single cell RNA sequencing, contained expanded highly-mutated IgA clones with an ovarian teratoma-restricted B cell population. Multiplex histology suggested tertiary lymphoid architectures in ovarian teratomas with dense B cell foci expressing the germinal centre marker BCL6, CD21+ follicular dendritic cells, and the NR1 subunit, alongside lymphatic vessels and high endothelial vasculature. Cultured teratoma explants and dissociated intratumoral B cells secreted NR1-IgGs in culture. Hence, ovarian teratomas showed structural and functional evidence of NR1-specific germinal centres. On exploring classical secondary lymphoid organs, B cells cultured from cervical lymph nodes of patients with NMDAR-antibody encephalitis produced NR1-IgG in 3/7 cultures, from patients with the highest serum NR1-IgG levels (P < 0.05). By contrast, NR1-IgG secretion was observed neither from cervical lymph nodes in disease controls nor in patients with adequately resected ovarian teratomas. Our multimodal evaluations provide convergent anatomical and functional evidence of NMDAR-autoantibody production from active germinal centres within both intratumoral tertiary lymphoid structures and traditional secondary lymphoid organs, the cervical lymph nodes. Furthermore, we develop a cervical lymph node sampling protocol that can be used to directly explore immune activity in health and disease at this emerging neuroimmune interface.

Syndromic surveillance of respiratory infections during protracted conflict: experiences from northern Syria 2016-2021.

OBJECTIVE: Northern Syria faces a large burden of influenza-like illness (ILI) and severe acute respiratory illness (SARI). This study aimed to investigate the trends of Early Warning and Response Network (EWARN) reported ILI and SARI in northern Syria between 2016 and 2021 and the potential impact of SARS-CoV-2. METHODS: We extracted weekly EWARN data on ILI/ SARI and aggregated cases and consultations into 4-week intervals to calculate case positivity. We conducted a seasonal-trend decomposition to assess case trends in the presence of seasonal fluctuations. RESULTS: It was observed that 4-week aggregates of ILI cases (n = 5,942,012), SARI cases (n = 114,939), ILI case positivity, and SARI case positivity exhibited seasonal fluctuations with peaks in the winter months. ILI and SARI cases in individuals aged ≥5 years surpassed those in individuals aged <5 years in late 2019. ILI cases clustered primarily in Aleppo and Idlib, whereas SARI cases clustered in Aleppo, Idlib, Deir Ezzor, and Hassakeh. SARI cases increased sharply in 2021, corresponding with a severe SARS-CoV-2 wave, compared with the steady increase in ILI cases over time. CONCLUSION: Respiratory infections cause widespread morbidity and mortality throughout northern Syria, particularly with the emergence of SARS-CoV-2. Strengthened surveillance and access to testing and treatment are critical to manage outbreaks among conflict-affected populations.

Cervical Cancer Screening by Refugee Category in a Refugee Health Primary Care Clinic in Calgary, Canada, 2011-2016.

Newly arrived refugees and refugee claimants experience low cervical cancer screening (CCS) rates in Canada. We investigated CCS at a dedicated refugee clinic. We completed a retrospective cohort study among patients at the Mosaic Refugee Health Clinic in Calgary, Canada, between 2011 and 2016. We investigated CCS offers and completion by refugee category. We then used multivariable logistic regression to estimate the association of CCS screening and refugee category, accounting for sociodemographic and clinical factors. We included 812 refugees. Most were married (71%) and had limited English proficiency (57%). Overall, 88% and 77% of patients were offered and completed screening, respectively. Compared to government assisted refugees, privately sponsored refugees completed CCS more often (OR 1.60, 95% CI [1.02-2.49]). A dedicated refugee clinic may provide effective CCS to newly arrived refugees irrespective of refugee category, insurance status or other barriers.

The impact of urban-based family medicine postgraduate rotations on rural preceptors/teachers.

BACKGROUND: The use of rural rotations within urban-based postgraduate programs is the predominant response of medical education to the health needs of underserved rural populations. The broader impact on rural physicians who teach has not been reported. METHODS: This study examined the personal, professional, and financial impact of a rural rotations for urban-based family medicine (UBFM) residents on Canadian rural teaching physicians. A survey was created and reviewed by community and academic rural physicians and a cohort of Canadian rural family physicians teaching UBFM residents was sampled. Survey data and free-text responses were assessed using quantitative and qualitative analyses. RESULTS: Participants with rural residency backgrounds perceived a negative impact of teaching UBFM (p = 0.02 personal and professional) and those in a primary rural environment (as defined below) perceived impact as positive (p < 0.001). Rural preceptors often held contrasting attitudes towards learners with negative judgements counter-balanced by positive thoughts. Duration in practice and of teaching experience did not have a significant impact on ratings. CONCLUSION: Being a rural preceptor of UBFM residents is rewarding but also stressful. The preceptor location of training and scope of practice appears to influence the impact of UBFM residents.

CONTEXTE: L'introduction de stages en milieu rural pour les résidents qui effectuent leur formation postdoctorale dans un centre urbain constitue la solution principale adoptée en matière d'éducation médicale pour répondre aux besoins des populations rurales mal desservies. L'impact plus large de ces stages sur les médecins enseignants en milieu rural n'a pas été documenté. MÉTHODES: Cette étude examine les répercussions personnelles, professionnelles et financières du stage réalisé en milieu rural par les résidents de médecine familiale en milieu urbain (MFMU) sur les médecins enseignants en milieu rural au Canada. Un sondage a été créé et revu par des médecins universitaires et communautaires en milieu rural et une cohorte de médecins de famille ruraux enseignant à des résidents de médecine familiale en milieu urbain a été échantillonnée. Les données du sondage et les réponses ouvertes obtenues ont fait l'objet d'analyses quantitative et qualitative. RÉSULTATS: Tandis que les participants possédant une expérience de résidence en milieu rural ont perçu l'effet négatif du fait d'enseigner aux résidents de MFMU (p = 0,02 personnel et professionnel), ceux qui exercent dans un environnement rural primaire (tel que défini ci-dessous) en ont une perception positive (p<0,001). Les superviseurs en milieu rural avaient souvent des attitudes contrastées envers les apprenants, des aspects positifs compensant certains jugements négatifs. La durée d'exercice et l'expérience en l'enseignement n'ont pas eu d'impact significatif sur les évaluations. CONCLUSION: Être un superviseur en milieu rural de résidents en MFMU est gratifiant, mais aussi stressant. Le lieu de formation et le champ d'exercice du superviseur semblent déterminer l'effet qu'ont les stages de résidents de MFMU sur ces superviseurs.

Weaponizing water as an instrument of war in Syria: Impact on diarrhoeal disease in Idlib and Aleppo governorates, 2011-2019.

OBJECTIVES: Investigate the weaponization of water during the Syrian conflict and the correlation of attacks on water, sanitation, and hygiene (WASH) infrastructure in Idlib and Aleppo governorates with trends in waterborne diseases reported by Early Warning and Response surveillance systems. METHODS: We reviewed literature and databases to obtain information on attacks on WASH in Aleppo and Idlib governorates between 2011 and 2019. We plotted weekly trends in waterborne diseases from two surveillance systems operational in Aleppo and Idlib governorates between 2015 and early 2020. RESULTS: The literature review noted several attacks on water and related infrastructure in both governorates, suggesting that WASH infrastructure was weaponized by state and non-state actors. Most interference with WASH in the Aleppo governorate occurred before 2019 and in the Idlib governorate in the summer of 2020. Other acute diarrhea represented >90% of cases of diarrhea; children under 5 years contributed 50% of cases. There was substantial evidence (p < 0.001) of an overall upward trend in cases of diarrheal disease. CONCLUSIONS: Though no direct correlation can be drawn between the weaponization of WASH and the burden of waterborne infections due to multiple confounders, this research introduces important concepts on attacks on WASH and their potential impacts on waterborne diseases.

Primary breast tumours but not lung metastases induce protective anti-tumour immune responses after Treg-depletion.

Although metastatic disease is responsible for the majority of cancer deaths, tests of novel immunotherapies in mouse tumour models often focus on primary tumours without determining whether these therapies also target metastatic disease. This study examined the impact of depleting Foxp3+ regulatory T cells (Treg), on lung metastases, using a mouse model of breast cancer. After Treg-depletion, generation of an immune response to the primary tumour was a critical determinant for limiting development of metastasis. Indeed, resection of the primary tumour abrogated any effect of Treg-depletion on metastases. In addition, whilst the immune response, generated by the primary tumour, prevented metastases development, it had little impact on controlling established disease. Collectively, the data indicate that metastatic cells in the lung are not controlled by immune responses induced by the primary tumour. These findings indicate that targeting Tregs alone will not suffice for treating lung metastases.

Detailed Molecular and Immune Marker Profiling of Archival Prostate Cancer Samples Reveals an Inverse Association between TMPRSS2:ERG Fusion Status and Immune Cell Infiltration.

Prostate cancer is a significant global health issue, and limitations to current patient management pathways often result in overtreatment or undertreatment. New ways to stratify patients are urgently needed. We conducted a feasibility study of such novel assessments, looking for associations between genomic changes and lymphocyte infiltration. An innovative workflow using an in-house targeted sequencing panel, immune cell profiling using an image analysis pipeline, RNA sequencing, and exome sequencing in select cases was tested. Gene fusions were profiled by RNA sequencing in 27 of 27 cases, and a significantly higher tumor-infiltrating lymphocyte (TIL) count was noted in tumors without a TMPRSS2:ERG fusion compared with those with the fusion (P = 0.01). Although this finding was not replicated in a larger validation set (n = 436) of The Cancer Genome Atlas images, there was a trend in the same direction. Differential expression analysis of TIL-high and TIL-low tumors revealed the enrichment of both innate and adaptive immune response pathways. Mutations in mismatch repair genes (MLH1 and MSH6 mutations in 1 of 27 cases) were identified. We describe a potential immune escape mechanism in TMPRSS2:ERG fusion-positive tumors. Detailed profiling, as shown herein, can provide novel insights into tumor biology. Likely differences with findings with other cohorts are related to methods used to define region of interest, but this warrants further study in a larger cohort.

Being a resident of family medicine in Africa in 2019: a picture from the 6th WONCA Africa Conference in Kampala / Ser residente em medicina de família na África em 2019: um retrato da sexta conferência WONCA África em Kampala / Ser residente en medicina familiar en África en 2019: un retrato de la sexta conferencia WONCA África en Kampala

Training young doctors in family medicine is challenging in any setting and many variables can influence the success or failure of a residency program. This article is the end result of a collaborative work that started in June 2019 at the WONCA Africa Regional Conference in Kampala, during a workshop lead by the Besrour Centre for Global Family Medicine at the College of Family Physicians of Canada. We present here the perspective of a small group of young African family physicians on the experience of being a resident in family medicine in Africa in 2019, hoping that the picture we depict here helps to promote the necessary improvements in the training programs in Africa for the near future.

Formar novos médicos de família é um desafio em qualquer cenário e muitas variáveis podem influenciar o sucesso ou o fracasso de um programa de residência. Este artigo é o resultado final de um trabalho colaborativo iniciado em junho de 2019, na Conferência Regional WONCA África em Kampala, Uganda, durante um workshop liderado pelo Centro Besrour do Colégio Canadense de Medicina de Família. Apresentamos aqui a perspectiva de um pequeno grupo de jovens médicos de família africanos sobre a experiência de ser residente em medicina de família na África em 2019, esperando que a imagem que representamos aqui ajude a promover as melhorias necessárias nos programas de residência na África em um futuro próximo.

La formación de nuevos médicos de familia es un desafío en cualquier lugar y muchas variables pueden influir en el éxito o el fracaso de un programa de residencia. Este artículo es el resultado final del trabajo colaborativo iniciado en junio de 2019 en la Conferencia Regional de África WONCA en Kampala, Uganda, durante un taller dirigido por el Centro Besrour del Colegio Canadiense de Medicina Familiar. Presentamos aquí la perspectiva de un pequeño grupo de jóvenes médicos de familia africanos sobre la experiencia de ser residente en medicina familiar en África en 2019, con la esperanza de que la imagen que representamos aquí ayude a promover mejoras en los programas de residencia en África en un futuro próximo.

Self-Maintaining CD103+ Cancer-Specific T Cells Are Highly Energetic with Rapid Cytotoxic and Effector Responses.

Enrichment of CD103+ tumor-infiltrating T lymphocytes (TIL) is associated with improved outcomes in patients. However, the characteristics of human CD103+ cytotoxic CD8+ T cells (CTL) and their role in tumor control remain unclear. We investigated the features and antitumor mechanisms of CD103+ CTLs by assessing T-cell receptor (TCR)-matched CD103+ and CD103- cancer-specific CTL immunity in vitro and its immunophenotype ex vivo Interestingly, we found that differentiated CD103+ cancer-specific CTLs expressed the active form of TGFß1 to continually self-regulate CD103 expression, without relying on external TGFß1-producing cells. The presence of CD103 on CTLs improved TCR antigen sensitivity, which enabled faster cancer recognition and rapid antitumor cytotoxicity. These CD103+ CTLs had elevated energetic potential and faster migration capacity. However, they had increased inhibitory receptor coexpression and elevated T-cell apoptosis following prolonged cancer exposure. Our data provide fundamental insights into the properties of matured human CD103+ cancer-specific CTLs, which could have important implications for future designs of tissue-localized cancer immunotherapy strategies.

Contrasting current challenges from the Brazilian and Canadian national health systems: The Besrour Papers: a series on the state of family medicine in Canada and Brazil.

OBJECTIVE: To compare the national health systems of Canada and Brazil and how both countries have addressed similar challenges in their primary care sectors. COMPOSITION OF THE COMMITTEE: A subgroup of the Besrour Centre of the College of Family Physicians of Canada developed connections with colleagues in Brazil and collaborated to undertake a between-country comparison, comparing and contrasting various elements of both countries' efforts to strengthen primary care over the past few decades. METHODS: Following a literature review, the authors collectively reflected on their experiences in an attempt to explore the past and current state of family medicine in Canada and Brazil. REPORT: The Brazilian and Canadian primary care systems are faced with similar challenges, including geography, demographic changes, population health inequities, and gaps in universal access to comprehensive primary care services. Although the approaches to addressing these challenges are different in both settings, they highlight the central importance of family physicians in both systems. Both countries continue to face considerable challenges in the context of mental health services in primary care. It remains important for Canada to draw lessons from the primary care systems and reforms of other countries, such as Brazil.

A global picture of family medicine: the view from a WONCA Storybooth.

BACKGROUND: Family Medicine is a novel discipline in many countries, where the motivation for training and value added to communities is not well-described. Our purpose was to understand the reason behind the choice of Family Medicine as a profession, the impact of Family Medicine on communities, and Family Medicine's characterizing qualities, as perceived by family doctors around the world. METHODS: One-question video interviews were conducted using an appreciative inquiry approach, with volunteer participants at the 2016 World Organization of Family Doctors conference in Rio de Janeiro. Qualitative data analysis applied the thematic, framework method. RESULTS: 135 family doctors from 55 countries participated in this study. Three overarching themes emerged: 1) key attributes of Family Medicine, 2) core Family Medicine values and 3) shared traits of family doctors. Family Medicine attributes and values were the key expressed motivators to join Family Medicine as a profession and were also among expressed factors that contributed to the impact of Family Medicine globally. Major sub-themes included the principles of comprehensive care, holistic care, continuity of care, patient centeredness, and the patient-provider relationship. Participants emphasized the importance of universal care, human rights, social justice and health equity. CONCLUSION: Family doctors around the world shared stories about their profession, presenting a heterogeneous picture of global Family Medicine unified by its attributes and values. These stories may inspire and serve as positive examples for Family Medicine programs, prospective students, advocates and other stakeholders.

Treg Depletion Licenses T Cell-Driven HEV Neogenesis and Promotes Tumor Destruction.

T-cell infiltration into tumors represents a critical bottleneck for immune-mediated control of cancer. We previously showed that this bottleneck can be overcome by depleting immunosuppressive Foxp3+ regulatory T cells (Tregs), a process that can increase frequencies of tumor-infiltrating lymphocytes through promoting the development of specialized portals for lymphocyte entry, namely high endothelial venules (HEVs). In this paper, we used a carcinogen-induced tumor model that allows for coevolution of the tumor microenvironment and the immune response to demonstrate that Treg depletion not only results in widespread disruption to HEV networks in lymph nodes (LNs) but also activates CD8+ T cells, which then drive intratumoral HEV development. Formation of these vessels contrasts with ontogenic HEV development in LNs in that the process is dependent on the TNF receptor and independent of lymphotoxin ß receptor-mediated signaling. These intratumoral HEVs do not express the chemokine CCL21, revealing a previously undescribed intratumoral blood vessel phenotype. We propose a model where Treg depletion enables a self-amplifying loop of T-cell activation, which promotes HEV development, T-cell infiltration, and ultimately, tumor destruction. The findings point to a need to test for HEV development as part of ongoing clinical studies in patients with cancer. Cancer Immunol Res; 5(11); 1005-15. ©2017 AACR.