Distinct Hodgkin lymphoma subtypes defined by noninvasive genomic profiling.

The scarcity of malignant Hodgkin and Reed-Sternberg cells hampers tissue-based comprehensive genomic profiling of classic Hodgkin lymphoma (cHL). By contrast, liquid biopsies show promise for molecular profiling of cHL due to relatively high circulating tumour DNA (ctDNA) levels1-4. Here we show that the plasma representation of mutations exceeds the bulk tumour representation in most cases, making cHL particularly amenable to noninvasive profiling. Leveraging single-cell transcriptional profiles of cHL tumours, we demonstrate Hodgkin and Reed-Sternberg ctDNA shedding to be shaped by DNASE1L3, whose increased tumour microenvironment-derived expression drives high ctDNA concentrations. Using this insight, we comprehensively profile 366 patients, revealing two distinct cHL genomic subtypes with characteristic clinical and prognostic correlates, as well as distinct transcriptional and immunological profiles. Furthermore, we identify a novel class of truncating IL4R mutations that are dependent on IL-13 signalling and therapeutically targetable with IL-4Rα-blocking antibodies. Finally, using PhasED-seq5, we demonstrate the clinical value of pretreatment and on-treatment ctDNA levels for longitudinally refining cHL risk prediction and for detection of radiographically occult minimal residual disease. Collectively, these results support the utility of noninvasive strategies for genotyping and dynamic monitoring of cHL, as well as capturing molecularly distinct subtypes with diagnostic, prognostic and therapeutic potential.

Tumor-associated antigen PRAME exhibits dualistic functions that are targetable in diffuse large B cell lymphoma.

PRAME is a prominent member of the cancer testis antigen family of proteins, which triggers autologous T cell-mediated immune responses. Integrative genomic analysis in diffuse large B cell lymphoma (DLBCL) uncovered recurrent and highly focal deletions of 22q11.22, including the PRAME gene, which were associated with poor outcome. PRAME-deleted tumors showed cytotoxic T cell immune escape and were associated with cold tumor microenvironments. In addition, PRAME downmodulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. In turn, PRC2-regulated genes were repressed in isogenic PRAME-KO lymphoma cell lines, and PRAME was found to directly interact with EZH2 as a negative regulator. EZH2 inhibition with EPZ-6438 abrogated these extrinsic and intrinsic effects, leading to PRAME expression and microenvironment restoration in vivo. Our data highlight multiple functions of PRAME during lymphomagenesis and provide a preclinical rationale for synergistic therapies combining epigenetic reprogramming with PRAME-targeted therapies.

The effect of smoking on chronic inflammation, immune function and blood cell composition.

Smoking is the number one risk factor for cancer mortality but only 15-20% of heavy smokers develop lung cancer. It would, therefore, be of great benefit to identify those at high risk early on so that preventative measures can be initiated. To investigate this, we evaluated the effects of smoking on inflammatory markers, innate and adaptive immune responses to bacterial and viral challenges and blood cell composition. We found that plasma samples from 30 heavy smokers (16 men and 14 women) had significantly higher CRP, fibrinogen, IL-6 and CEA levels than 36 non-smoking controls. Whole blood samples from smokers, incubated for 7 h at 37 °C in the absence of any exogenous stimuli, secreted significantly higher levels of IL-8 and a number of other cytokines/chemokines than non-smokers. When challenged for 7 h with E. coli, whole blood samples from smokers secreted significantly lower levels of many inflammatory cytokines/chemokines. However, when stimulated with HSV-1, significantly higher levels of both PGE2 and many cytokines/chemokines were secreted from smokers' blood samples than from controls. In terms of blood cell composition, red blood cells, hematocrits, hemoglobin levels, MCV, MCH, MCHC, Pct and RDW levels were all elevated in smokers, in keeping with their compromised lung capacity. As well, total leukocytes were significantly higher, driven by increases in granulocytes and monocytes. In addition, smokers had lower NK cells and higher Tregs than controls, suggesting that smoking may reduce the ability to kill nascent tumor cells. Importantly, there was substantial person-to person variation amongst smokers with some showing markedly different values from controls and others showing normal levels of many parameters measured, indicating the former may be at significantly higher risk of developing lung cancer.

Exploratory examination of inflammation state, immune response and blood cell composition in a human obese cohort to identify potential markers predicting cancer risk.

Obesity has reached epidemic proportions and is often accompanied by elevated levels of pro-inflammatory cytokines that promote many chronic diseases, including cancer. However, not all obese people develop these diseases and it would be very helpful to identify those at high risk early on so that preventative measures can be instituted. We performed an extensive evaluation of the effects of obesity on inflammatory markers, on innate and adaptive immune responses, and on blood cell composition to identify markers that might be useful in distinguishing those at elevated risk of cancer. Plasma samples from 42 volunteers with a BMI>35 had significantly higher CRP, PGE2, IL-1RA, IL-6 and IL-17 levels than 34 volunteers with normal BMIs. Of the cytokines and chemokines tested, only IL-17 was significantly higher in men with a BMI>35 than women with a BMI>35. As well, only IL-17 was significantly higher in those with a BMI>35 that had type 2 diabetes versus those without type 2 diabetes. Whole blood samples from participants with a BMI>35, when challenged with E. coli, produced significantly higher levels of IL-1RA while HSV-1 challenge resulted in significantly elevated IL-1RA and VEGF, and a non-significant increase in G-CSF and IL-8 levels. T cell activation of PBMCs, via anti-CD3 plus anti-CD28, resulted in significantly higher IFNγ production from volunteers with a BMI>35. In terms of blood cells, red blood cell distribution width (RDW), monocytes, granulocytes, CD4+T cells and Tregs were all significantly higher while, natural killer (NK) and CD8+ T cells were all significantly lower in the BMI>35 cohort, suggesting that obesity may reduce the ability to kill nascent tumor cells. Importantly, however, there was considerable person-to-person variation amongst participants with a BMI>35, with some volunteers showing markedly different values from controls and others showing normal levels of many parameters measured. These person-to-person variations may prove useful in identifying those at high risk of developing cancer.

The effect of diet and exercise on tobacco carcinogen-induced lung cancer.

In previous studies, we found that low-carbohydrate (CHO) diets reduced the incidence of tumors in mice genetically predisposed to cancer. However, because >90% of human cancers arise via carcinogen-induced somatic mutations, we investigated, herein, the role that different types and levels of CHO, protein and lipid play in lung cancer induced by the tobacco-specific carcinogen, nicotine-derived nitrosamine ketone (NNK) in A/J mice. We found lowering CHO levels significantly reduced lung nodules and blood glucose levels. We also found that soy protein was superior to casein and that coconut oil was ineffective at reducing lung nodules. Diets containing amylose or inulin (at 15% of total calories), soy protein (at 35%) and fat (at 50%, 30% being fish oil) were the most effective at reducing lung nodules. These fish oil-containing diets increased plasma levels of the ketone body, ß-hydroxybutyrate, while reducing both insulin and 8-isoprostane in plasma and bronchoalveolar interleukin-12 and lung PGE2 levels. After only 2 weeks on this diet, the levels of γ-H2AX were significantly reduced, 24 hours after NNK treatment. Housing these mice in two-tiered rat cages with exercise wheels led to similar mouse weights on the different diets, whereas keeping mice in standard mouse cages led to both significant weight differences between the low-CHO, soy protein, fish oil diet and Western diet and substantially more lung nodules than in the two-tiered cages. Our results suggest that low-CHO, soy protein, fish oil-containing diets, together with exercise, may reduce the incidence of lung cancer.

Effect of age on chronic inflammation and responsiveness to bacterial and viral challenges.

To identify reliable biomarkers of age-related changes in chronic inflammation and responsiveness to bacterial and viral challenges, we evaluated endogenous and ex vivo stimulated levels of 18 inflammatory markers, using whole blood collected in EDTA and sodium heparin tubes from 41 healthy volunteers, i.e., 11 men + 10 women aged 20-35 and 10 men + 10 women aged 50-77. These studies revealed significant differences in the levels of inflammatory markers when blood was collected in EDTA versus sodium heparin and age related differences in these biomarkers were confirmed with blood collected in EDTA from 120 healthy volunteers in 3 age categories, ie, 20 men + 20 women, aged 20-35, 36-49 and 50-77. Studies with unstimulated blood samples, to measure levels of chronic inflammation, revealed a significant increase with age in IL-12p70, CRP and PGE2, consistent with the concept of "inflammaging", and a decrease in G-CSF in both men and women. Interestingly, in response to E. coli stimulation, PGE2 levels were markedly reduced in the 50-77 year old cohort while they were increased following Herpes Simplex virus-1 (HSV-1) stimulation, along with IL-8. In addition, unlike E. coli, HSV-1 potently stimulated IFNα production, but levels were dramatically reduced in the older cohort, consistent with a reduced ability to generate an anti-viral response. We also found platelets and CD8+ T cells were reduced with age while CD4+ T cells were significantly increased, resulting in a substantially higher CD4/CD8 ratio in the older cohort. Surprisingly, however, we found that the older cohort exhibited more T cell proliferation and IFNγ production in response to anti-CD3+anti-CD28 stimulation. Importantly, there was considerable person-to-person variation in these inflammatory markers in all age groups, making possible comparisons between a person's "inflammage" and chronological age. These assays should help to identify individuals at high risk of autoimmune disorders and cancer.