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MTS assays on cell lines after treatment with ADCT-301, Non-binding ADC and naked antibody, HuMax-TAC

Assessing APOBEC3 gene expression using immunohistochemical and bioinformatic approaches.

Immune cell deconvolution from molecular data. Relative proportions of immune cell subtypes are predicted from gene expression data using the Danaher method, and from methylation data using methylCIBERSORT. Differences between progressive and regressive groups are reported here.

Despite the many advances in the treatment of hematologic malignancies over the past decade, outcomes in refractory lymphomas remain poor. One potential strategy in this patient population is the specific targeting of IL2R-α (CD25), which is overexpressed on many lymphoma and leukemic cells, using antibody–drug conjugates (ADC). ADCT-301 is an ADC composed of human IgG1 HuMax-TAC against CD25, stochastically conjugated through a dipeptide cleavable linker to a pyrrolobenzodiazepine (PBD) dimer warhead with a drug–antibody ratio (DAR) of 2.3. ADCT-301 binds human CD25 with picomolar affinity. ADCT-301 has highly potent and selective cytotoxicity against a panel of CD25-expressing human lymphoma cell lines. Once internalized, the released warhead binds in the DNA minor groove and exerts its potent cytotoxic action via the formation of DNA interstrand cross-links. A strong correlation between loss of viability and DNA cross-link formation is demonstrated. DNA damage persists, resulting in phosphorylation of histone H2AX, cell-cycle arrest in G2–M, and apoptosis. Bystander killing of CD25-negative cells by ADCT-301 is also observed. In vivo, a single dose of ADCT-301 results in dose-dependent and targeted antitumor activity against both subcutaneous and disseminated CD25-positive lymphoma models. In xenografts of Karpas 299, which expressed both CD25 and CD30, marked superiority over brentuximab vedotin (Adcetris) is observed. Dose-dependent increases in DNA cross-linking, γ-H2AX, and PBD payload staining were observed in tumors in vivo indicating a role as relevant pharmacodynamic assays. Together, these data support the clinical testing of this novel ADC in patients with CD25-expressing tumors. Mol Cancer Ther; 15(11); 2709–21. ©2016 AACR.

APOBEC3 gene expression during senescence and cell cycle progression.

In vivo efficacy in Karpas disseminated models. Kaplan Meier survival plots of disseminated Karpas 299 models

Comet assay on CD25-negative Daudi cells after ADCT-301 and SG3199 treatment

Supplementary Methods

In vivo PBMC DNA cross-linking. DNA cross-linking as measured in CD25-negative PBMCs from SCID mice treated with ADCT-301

DAR determination by SEC, RPLC and HIC

Supplementary Table 1: Overview of data resource. Supplementary Table 2: List of antibodies used for immunofluorescence. Supplementary Table 3: List of antibodies used for western blotting. Supplementary Table 4. List of primers used for PCR.

Generation and characterization of A3A and A3B knockouts in TIIP cells.

A complete list of investigators in the Tracking Non-Small Cell Lung Cancer Evolution through Therapy (TRACERx) Consortium

Correlation between APOBEC3 expression and different measures of CIN.

In vivo efficacy in Karpas 299 subcutaneous models. Kaplan Meier survival plots of subcutaneous models in Figures 5A and 5B

APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when APOBEC3 expression is induced during cancer development remains to be defined. Here we show that specific APOBEC3 genes are upregulated in breast ductal carcinoma in situ, and in preinvasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx preinvasive to invasive non–small cell lung cancer (NSCLC) lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G1 phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models revealed APOBEC3B expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in preinvasive disease, providing fuel for selection early in cancer evolution.Significance:This study reveals the dynamics and drivers of APOBEC3 gene expression in preinvasive disease and the exacerbation of cellular diversity by APOBEC3B through DNA replication stress to promote chromosomal instability early in cancer evolution.This article is highlighted in the In This Issue feature, p. 2355

In vivo efficacy in Ramos disseminated model. Kaplan Meier survival plots of disseminated CD25-negative Ramos model

Before squamous cell lung cancer develops, precancerous lesions can be found in the airways. From longitudinal monitoring, we know that only half of such lesions become cancer, whereas a third spontaneously regress. Although recent studies have described the presence of an active immune response in high-grade lesions, the mechanisms underpinning clinical regression of precancerous lesions remain unknown. Here, we show that host immune surveillance is strongly implicated in lesion regression. Using bronchoscopic biopsies from human subjects, we find that regressive carcinoma in situ lesions harbor more infiltrating immune cells than those that progress to cancer. Moreover, molecular profiling of these lesions identifies potential immune escape mechanisms specifically in those that progress to cancer: antigen presentation is impaired by genomic and epigenetic changes, CCL27–CCR10 signaling is upregulated, and the immunomodulator TNFSF9 is downregulated. Changes appear intrinsic to the carcinoma in situ lesions, as the adjacent stroma of progressive and regressive lesions are transcriptomically similar.Significance:Immune evasion is a hallmark of cancer. For the first time, this study identifies mechanisms by which precancerous lesions evade immune detection during the earliest stages of carcinogenesis and forms a basis for new therapeutic strategies that treat or prevent early-stage lung cancer.See related commentary by Krysan et al., p. 1442.This article is highlighted in the In This Issue feature, p. 1426

Supplementary Data from Systematic Evaluation of the Immune Environment of Small Intestinal Neuroendocrine Tumors

Purpose:The immune tumor microenvironment and the potential therapeutic opportunities for immunotherapy in small intestinal neuroendocrine tumors (siNET) have not been fully defined.Experimental Design:Herein, we studied 40 patients with primary and synchronous metastatic siNETs, and matched blood and normal tissue obtained during surgery. We interrogated the immune checkpoint landscape using multi-parametric flow cytometry. In addition, matched FFPE tissue was obtained for multi-parametric IHC to determine the relative abundance and distribution of T-cell infiltrate. Tumor mutational burden (TMB) was also assessed and correlated with immune infiltration.Results:Effector tumor-infiltrating lymphocytes (TIL) had a higher expression of PD-1 in the tumor microenvironment compared with the periphery. In addition, CD8+ TILs had a significantly higher co-expression of PD-1/ICOS and PD-1/CTLA-4 (cytotoxic T lymphocyte antigen-4) and higher levels of PD-1 expression compared with normal tissue. IHC revealed that the majority of cases have ≤10% intra-tumoral T cells but a higher number of peri-tumoral T cells, demonstrating an “exclusion” phenotype. Finally, we confirmed that siNETs have a low TMB compared with other tumor types in the TCGA database but did not find a correlation between TMB and CD8/Treg ratio.Conclusions:Taken together, these results suggest that a combination therapy approach will be required to enhance the immune response, using PD-1 as a checkpoint immunomodulator backbone in combination with other checkpoint targeting molecules (CTLA-4 or ICOS), or with drugs targeting other pathways to recruit “excluded” T cells into the tumor microenvironment to treat patients with siNETs.

Supplementary Table from Systematic Evaluation of the Immune Environment of Small Intestinal Neuroendocrine Tumors

The spatial architecture of the lymphoid tissue in follicular lymphoma (FL) presents unique challenges to studying its immune microenvironment. We investigated the spatial interplay of T cells, macrophages, myeloid cells and natural killer T cells using multispectral immunofluorescence images of diagnostic biopsies of 32 patients. A deep learning-based image analysis pipeline was tailored to the needs of follicular lymphoma spatial histology research, enabling the identification of different immune cells within and outside neoplastic follicles. We analyzed the density and spatial co-localization of immune cells in the inter-follicular and intra-follicular regions of follicular lymphoma. Low inter-follicular density of CD8+FOXP3+ cells and co-localization of CD8+FOXP3+ with CD4+CD8+ cells were significantly associated with relapse (p = 0.0057 and p = 0.0019, respectively) and shorter time to progression after first-line treatment (Logrank p = 0.0097 and log-rank p = 0.0093, respectively). A low inter-follicular density of CD8+FOXP3+ cells is associated with increased risk of relapse independent of follicular lymphoma international prognostic index (FLIPI) (p = 0.038, Hazard ratio (HR) = 0.42 [0.19, 0.95], but not independent of co-localization of CD8+FOXP3+ with CD4+CD8+ cells (p = 0.43). Co-localization of CD8+FOXP3+ with CD4+CD8+ cells is predictors of time to relapse independent of the FLIPI score and density of CD8+FOXP3+ cells (p = 0.027, HR = 0.0019 [7.19 × 10

Patient-derived xenograft (PDX) models involve the engraftment of tumour tissue in immunocompromised mice and represent an important pre-clinixtcal oncology research. A limitation of non-small cell lung cancer (NSCLC) PDX model derivation in NOD-scidIL2Rgammanull(NSG) mice is that a subset of initial engraftments are of lymphocytic, rather than tumour origin. In the lung TRACERx PDX pipeline, lymphoproliferations occurred in 17.8% of lung adenocarcinoma and 10% of lung squamous cell carcinoma transplantations, despite none of these patients having a prior or subsequent clinical history of lymphoproliferative disease. Lymphoproliferations were predominantly human CD20+ B cells and had the immunophenotype expected for post-transplantation diffuse large B cell lymphoma. All lymphoproliferations expressed Epstein-Barr-encoded RNAs (EBER). Analysis of immunoglobulin light chain gene rearrangements in three tumours where multiple tumour regions had resulted in lymphoproliferations suggested that each had independent clonal origins. Overall, these data suggest the presence of B cell clones with lymphoproliferative potential within primary NSCLC tumours that are under continuous immune surveillance. Since these cells can be expanded following transplantation into NSG mice, our data highlight the value of quality control measures to identify lymphoproliferations within xenograft pipelines and support the incorporation of strategies to minimise lymphoproliferations during the early stages of xenograft establishment pipelines. To present the histology data herein, we developed a Python-based tool for generating patient-level pathology overview figures from whole-slide image files; PATHOverview is available on GitHub (https://github.com/EpiCENTR-Lab/PATHOverview).

Background & Aims HIV-seropositivity shortens survival in patients with hepatocellular carcinoma (HCC). Whilst risk factors for HCC including Hepatitis C virus infection can influence T-cell phenotype, it is unknown whether HIV can influence functional characteristics of the T-cell infiltrate. Methods From the Liver Cancer in HIV biorepository, we derived 129 samples of transplanted (76%) or resected (20%) HCC in 8 European and North American centres. We profiled intra and peri-tumoural tissue to evaluate regulatory CD4+/FOXP3+ and immune-exhausted CD8+/PD1+ T-cells in HIV+ (n=66) and HIV- (n=63) samples. We performed targeted transcriptomics and T-cell receptor sequencing in a restricted subset of samples evaluated in relationship with HIV status. We correlated immuno-pathologic features with patients’ characteristics including markers of HIV infection. Results Of the 66 HIV+ patients, 83% were Hepatitis C virus co-infected with an undetectable HIV viral load (51%) and a median blood CD4+ cell count of 430 cells/mm3 (range 15-908). HIV+ patients were compared with HIV- controls with similar staging characteristics including Barcelona Clinic Liver Cancer (BCLC) stage A-B (86% vs. 83%, p=0.16)

Aims Burkitt lymphoma (BL) is an aggressive B-cell lymphoma that, in some instances, may show a granulomatous reaction associated with a favourable prognosis and occasional spontaneous regression. In the present study, we aimed to define the tumour microenvironment (TME) in four such cases, two of which regressed spontaneously. Methods and results All cases showed aggregates of tumour cells with the typical morphology, molecular cytogenetics and immunophenotype of BL surrounded by a florid epithelioid granulomatous reaction. All four cases were Epstein-Barr virus (EBV)-positive with type I latency. Investigation of the TME showed similar features in all four cases. The analysis revealed a proinflammatory response triggered by Th1 lymphocytes and M1 polarised macrophages encircling the neoplastic cells with a peculiar topographic distribution. Conclusions Our data provide an in-vivo picture of the role that specific immune cell subsets might play during the early phase of BL, which may be capable of maintaining the tumour in a self-limited state or inducing its regression. These novel results may provide insights into new potential therapeutic avenues in EBV-positive BL patients in the era of cellular immunotherapy.

Funder: CUH | Addenbrooke’s Charitable Trust, Cambridge University Hospitals (Addenbrooke’s Charitable Trust, Cambridge University Hospitals NHS Foundation Trust); doi: https://doi.org/10.13039/501100002927, Funder: Pathological Society of Great Britain and Ireland; doi: https://doi.org/10.13039/501100000672, The development of extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) is driven by chronic inflammatory responses and acquired genetic changes. To investigate its genetic bases, we performed targeted sequencing of 93 genes in 131 MALT lymphomas including 76 from the thyroid. We found frequent deleterious mutations of TET2 (86%), CD274 (53%), TNFRSF14 (53%), and TNFAIP3 (30%) in thyroid MALT lymphoma. CD274 was also frequently deleted, together with mutation seen in 68% of cases. There was a significant association between CD274 mutation/deletion and TNFRSF14 mutation (p = 0.001). CD274 (PD-L1) and TNFRSF14 are ligands for the co-inhibitory receptor PD1 and BTLA on T-helper cells, respectively, their inactivation may free T-cell activities, promoting their help to malignant B-cells. In support of this, both the proportion of activated T-cells (CD4+CD69+/CD4+) within the proximity of malignant B-cells, and the level of transformed blasts were significantly higher in cases with CD274/TNFRSF14 genetic abnormalities than those without these changes. Both CD274 and TNFRSF14 genetic changes were significantly associated with Hashimoto's thyroiditis (p = 0.01, p = 0.04, respectively), and CD274 mutation/deletion additionally associated with increased erythrocyte sedimentation rate (p = 0.0001). In conclusion, CD274/TNFRSF14 inactivation in thyroid MALT lymphoma B-cells may deregulate their interaction with T-cells, promoting co-stimulations and impairing peripheral tolerance.

Purpose: The immune tumor microenvironment and the potential therapeutic opportunities for immunotherapy in small intestinal neuroendocrine tumors (siNET) have not been fully defined. Experimental Design: Herein, we studied 40 patients with primary and synchronous metastatic siNETs, and matched blood and normal tissue obtained during surgery. We interrogated the immune checkpoint landscape using multi-parametric flow cytometry. In addition, matched FFPE tissue was obtained for multi-parametric IHC to determine the relative abundance and distribution of T-cell infiltrate. Tumor mutational burden (TMB) was also assessed and correlated with immune infiltration. Results: Effector tumor-infiltrating lymphocytes (TIL) had a higher expression of PD-1 in the tumor microenvironment compared with the periphery. In addition, CD8+ TILs had a significantly higher co-expression of PD-1/ICOS and PD-1/CTLA-4 (cytotoxic T lymphocyte antigen-4) and higher levels of PD-1 expression compared with normal tissue. IHC revealed that the majority of cases have ≤10% intra-tumoral T cells but a higher number of peri-tumoral T cells, demonstrating an “exclusion” phenotype. Finally, we confirmed that siNETs have a low TMB compared with other tumor types in the TCGA database but did not find a correlation between TMB and CD8/Treg ratio. Conclusions: Taken together, these results suggest that a combination therapy approach will be required to enhance the immune response, using PD-1 as a checkpoint immunomodulator backbone in combination with other checkpoint targeting molecules (CTLA-4 or ICOS), or with drugs targeting other pathways to recruit “excluded” T cells into the tumor microenvironment to treat patients with siNETs.

Immune-related hepatitis is an important toxicity from immune-checkpoint inhibitor therapy, affecting up to 20% of patients on dual cytotoxic T-lymphocyte antigen 4/programmed cell death 1 (CTLA-4/PD-1) inhibitors. The mechanisms underlying this type of drug-induced liver injury are poorly understood. We report the case of a patient with immune-checkpoint inhibitor-related hepatitis where the presence of a diffuse granulomatous, PD-L1-positive infiltrate on liver biopsy correlated with poor response to corticosteroids. Our findings suggest a potential role for activation of the PD-1 pathway within the histiocitic infiltrate as a mechanism of toxicity. Further studies should address the role of macrophages in this patient group characterized by steroid-refractoriness.

Patient-derived xenograft (PDX) models have become important models in cancer biology. They are thought to mimic tumor biology more closely than traditional cell lines as a consequence of their in vivo heterocellularity and cell-matrix interactions, their 3D architecture and their relatively recent derivation. The genomic fidelity of PDXs is integral to their applications in both basic research and in translational medicine. We derived multi-region PDX models from patients enrolled in TRACERx - a study of the evolutionary dynamics of non-small cell lung cancer (NSCLC) that uses a multi-region deep whole-exome sequencing (WES) approach - using more extensive spatial sampling to better understand the histological and genomic fidelity of the PDX approach. We transplanted regional NSCLC tumor tissue subcutaneously into immunocompromised NSG mice. 134 regions were attempted, resulting in 60 passagable xenografts (44.8%). Of these, 44 regional xenografts (32.8% of all regions) were NSCLC-derived, while 16 (11.9% of all regions) were B-cell lymphoproliferative disease. Multi-region sampling revealed heterogeneous success of PDX derivation between regions within individual patient tumors. There was correlation between PDX derivation and tumor purity by WES. Overall, NSCLC PDX models were established for 22 patients (50%; range 1-5 regional models per patient; Table 1). Histologically, broad similarity was observed between PDX models and corresponding patient tumor regions, although in a small number of models, variability was noted between the patient tumor and the corresponding PDX model, or during PDX passaging. PDXs were subjected to WES for comparison with matched patient tumor regions. Overall, our study demonstrates the feasibility of systematic multi-region PDX derivation and suggests that multiple spatial sampling of tumors could improve PDX take rates and generate PDXs that represent the intratumor diversity of heterogenous NSCLCs. Table 1. Engraftment rates of multiple spatial regions from non-small cell lung cancer tumors. Attempted NSCLC PDX B-Lymphoproliferations No Xenograft: Regions No Xenograft: Any Region LUAD 48 (21) 12 (7) 9 (7) 27 (10) LUSC 61 (17) 20 (11) 6 (5) 35 (4) Other 25 (6) 12 (4) 1 (1) 12 (1) TOTAL 134 (44) 44 (22) 16 (13) 74 (15) Citation Format: Robert E. Hynds, David R. Pearce, Ayse U. Akarca, Sophia Ward, Ariana Huebner, Oriol Pich, Gareth A. Wilson, Kate H. Gowers, Rebecca Towns, Assma Ben Aissa, Selvaraju Veeriah, Sergio A. Quezada, Mariam Jamal-Hanjani, Sam M. Janes, Nicholas McGranahan, David A. Moore, Teresa Marafioti, Charles Swanton. Multi-region patient-derived xenograft models from non-small cell lung cancer patients enrolled in lung TRACERx [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3096.

Intratumoral regulatory T cell (Treg) abundance associates with diminished anti-tumor immunity and poor prognosis in human cancers. Recent work demonstrates that CD25, the high affinity receptor subunit for IL-2, is a selective target for Treg depletion in mouse and human malignancies; however, anti-human CD25 antibodies have failed to deliver clinical responses against solid tumors due to bystander IL-2 receptor signaling blockade on effector T cells, which limits their anti-tumor activity. Here we demonstrate potent single-agent activity of anti-CD25 antibodies optimized to deplete Tregs whilst preserving IL-2-STAT5 signaling on effector T cells, and demonstrate synergy with immune checkpoint blockade in vivo. Pre-clinical evaluation of an anti-human CD25 (RG6292) antibody with equivalent features demonstrates, in both non-human primates and humanized mouse models, efficient Treg depletion with no overt immune-related toxicities. Our data supports the clinical development of RG6292 and evaluation of novel combination therapies incorporating non-IL-2 blocking anti-CD25 antibodies in clinical studies.

BackgroundEndogenous retroviruses (ERVs) play a role in a variety of biological processes, including embryogenesis and cancer. DNA methyltransferase inhibitors (DNMTi)-induced ERV expression triggers interferon responses in ovarian cancer cells via the viral sensing machinery. Baseline expression of ERVs also occurs in cancer cells, though this process is poorly understood and previously unexplored in epithelial ovarian cancer (EOC). Here, the prognostic and immunomodulatory consequences of baseline ERV expression was assessed in EOC.MethodsERV expression was assessed using EOC transcriptional data from The Cancer Genome Atlas (TCGA) and from an independent cohort (Hammersmith Hospital, HH), as well as from untreated or DNMTi-treated EOC cell lines. Least absolute shrinkage and selection operator (LASSO) logistic regression defined an ERV expression score to predict patient prognosis. Immunohistochemistry (IHC) was conducted on the HH cohort. Combination of DNMTi treatment with γδ T cells was tested in vitro, using EOC cell lines and patient-derived tumor cells.ResultsERV expression was found to define clinically relevant subsets of EOC patients. An ERV prognostic score was successfully generated in TCGA and validated in the independent cohort. In EOC patients from this cohort, a high ERV score was associated with better survival (log-rank p=0.0009) and correlated with infiltration of CD8+PD1+T cells (r=0.46, p=0.0001). In the TCGA dataset, a higher ERV score was found in BRCA1/2 mutant tumors, compared to wild type (p=0.015), while a lower ERV score was found in CCNE1 amplified tumors, compared to wild type (p=0.019). In vitro, baseline ERV expression dictates the level of ERV induction in response to DNMTi. Manipulation of an ERV expression threshold by DNMTi resulted in improved EOC cell killing by cytotoxic immune cells.ConclusionsThese findings uncover the potential for baseline ERV expression to robustly inform EOC patient prognosis, influence tumor immune infiltration and affect antitumor immunity.

The reprogramming of a patient’s immune system through genetic modification of the T cell compartment with chimeric antigen receptors (CARs) has led to durable remissions in chemotherapy-refractory B cell cancers. Targeting of solid cancers by CAR-T cells is dependent on their infiltration and expansion within the tumor microenvironment, and thus far, fewer clinical responses have been reported. Here, we report a phase 1 study (NCT02761915) in which we treated 12 children with relapsed/refractory neuroblastoma with escalating doses of second-generation GD2-directed CAR-T cells and increasing intensity of preparative lymphodepletion. Overall, no patients had objective clinical response at the evaluation point +28 days after CAR-T cell infusion using standard radiological response criteria. However, of the six patients receiving ≥108/meter2 CAR-T cells after fludarabine/cyclophosphamide conditioning, two experienced grade 2 to 3 cytokine release syndrome, and three demonstrated regression of soft tissue and bone marrow disease. This clinical activity was achieved without on-target off-tumor toxicity. Targeting neuroblastoma with GD2 CAR-T cells appears to be a valid and safe strategy but requires further modification to promote CAR-T cell longevity.

An amendment to this paper has been published and can be accessed via the original article.

Background The Tumor Microenviroment (TME) is a complex milieu that is increasingly recognized as a key factor in multiple stages of disease progression and responses to therapy as well as escape from immune surveillance. However, the precise contribution of specific immune effector and immune suppressor components of the TME in Burkitt lymphoma (BL) remains poorly understood. Methods In this paper, we applied the computational algorithm CIBERSORT to Gene Expression Profiling (GEP) datasets of 40 BL samples to draw a map of immune and stromal components of TME. Furthermore, by multiple immunohistochemistry (IHC) and multispectral immunofluorescence (IF), we investigated the TME of additional series of 40 BL cases to evaluate the role of the Programmed Death-1 and Programmed Death Ligand-1 (PD-1/PD-L1) immune checkpoint axis. Results Our results indicate that M2 polarized macrophages are the most prominent TME component in BL. In addition, we investigated the correlation between PD-L1 and latent membrane protein-2A (LMP2A) expression on tumour cells, highlighting a subgroup of BL cases characterized by a non-canonical latency program of EBV with an activated PD-L1 pathway. Conclusion In conclusion, our study analysed the TME in BL and identified a tolerogenic immune signature highlighting new potential therapeutic targets.

APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when APOBEC3 expression is induced during cancer development remains to be defined. Here we show that specific APOBEC3 genes are upregulated in breast ductal carcinoma in situ, and in preinvasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx preinvasive to invasive non–small cell lung cancer (NSCLC) lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G1 phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models revealed APOBEC3B expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in preinvasive disease, providing fuel for selection early in cancer evolution. Significance: This study reveals the dynamics and drivers of APOBEC3 gene expression in preinvasive disease and the exacerbation of cellular diversity by APOBEC3B through DNA replication stress to promote chromosomal instability early in cancer evolution. This article is highlighted in the In This Issue feature, p. 2355

SummaryThe discovery of mutated Calreticulin (CALR) in myeloproliferative neoplasms (MPN) has provided proof of clonality, diagnostic importance, and influence on prognosis of this pathology. The identification of this MPN-associated driver mutation -currently based on molecular assays- is represented as a major diagnostic criterion for essential thrombocythaemia (ET), pre-fibrotic myelofibrosis and primary myelofibrosis (PMF) in the updated World Health Organization (WHO) 2008 classification. In the present study, we validated by immunohistochemistry the diagnostic usefulness of the monoclonal CAL2 antibody. Cases of acute myeloid leukaemia (AML) and myelodysplastic/ myeloproliferative neoplasms (MDS/MPN) have been also investigated to assess the specificity of CAL2 antibody. For this purpose, the result of the CAL2 immunostaining was compared with the result of molecular assays. Additionally, we investigated by double staining whether expression of mutated CALR can also be demonstrated on cells of the erythroid and myeloid lineage. We confirmed the usefulness of the CAL2 monoclonal antibody in successfully detecting mutant CALR in bone marrow biopsies. We showed that the immune-reactivity of CAL2 was absolutely restricted to the presence of CALR mutations, which were seen only in ET and MDS/MPN biopsies, but not in AML biopsies (14/14). There was 100% concordance in biopsy specimens with the concomitant molecular results. We applied double staining technique and confirmed that a subpopulation of granulopoietic and erythropoietic cells express mutated CALR as demonstrated with the CAL2 antibody in cases of MPNs. This supports the suggestion that the CALR mutations occur in a multipotent progenitor capable of generating both myeloid and erythroid progeny with preferential expansion of megakaryocytic cell lineage as a result of CALR mutation in an immature hematopoietic stem cell.

Background and AimsT lymphocytes are found in abnormally large numbers in the bowel in Crohn’s disease. This has led to the assumption by some that these cells play a causal role in the pathogenesis of what has been labelled an autoimmune disease. An alternative explanation for their presence is that, as part of the adaptive immune system, the accumulation of these cells is not a primary phenomenon, but is a secondary adaptive immune response to faecal material in the bowel wall. To distinguish between these two processes we compared the T-cell repertoire in the bowel in Crohn’s with that in diverticulitis, where the primary pathology is mechanical, with a subsequent immune response to the accumulated faecal material.MethodsSix cases of Crohn’s disease and six patients with diverticulitis were studied. Dewaxed sections of bowel were stained with Anti-CD4, Anti-CD8, Anti-FOXP3 and Anti-CD25 to identify cytotoxic T-cells, NK-Tcells; T-helper and T-reg T-cells.ResultsNo differences were found in the distribution of the different T-cell markers in either the mucosa or in areas of inflammation in the two conditions.ConclusionThe accumulation of T-lymphocytes in the bowel in Crohn’s disease is likely to be a sign of an adaptive immune response to faecal material within the bowel rather than an indication of a primary causal immune attack on the bowel that produces the disease.

Immunotherapy; Liver neoplasms Inmunoterapia; Neoplasias hepáticas Immunoteràpia; Neoplàsies hepàtiques Background 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

Background: Responses to checkpoint inhibitor (CPI) monotherapy in patients with metastatic castration resistant prostate cancer (mCRPC) have been limited. This is likely due to a “cold” tumour immune microenvironment. We hypothesised that patients with mCRPC will be more likely to respond if they have a positive immunogenic signature (ImS+). We report the response/safety for NIVO + IPI in pts with ImS+ mCRPC from cohort 1 of the NEPTUNES study. Methods: Pts with mCRPC who progressed following ≥1 line of therapy and ImS+ were eligible. ImS+ was defined by ≥1 of the following: 1) mismatch repair deficient (MMRD) by immunohistochemistry (IHC); 2) DNA damage repair deficient (DDRD) excluding MMRD, detected by the UW-OncoPlex targeted exome sequencing assay and; 3) high tumour infiltrating lymphocytes (TILs) on multiplexed immunohistochemistry (CD4, CD8 or FoxP3+ >20% nucleated cells). Treatment was NIVO 1 mg/kg + IPI 3 mg/kg Q3W for 4 doses, then NIVO 480 mg every 4 weeks. Primary endpoint was composite response rate (CRR) defined by ≥1 of the following: 1) radiological response by RECIST 1.1; 2) PSA response ≥50%; 3) conversion of circulating tumour cells (CTC) at week 9. Treatment would be deemed ineffective if the CRR was Conclusions: NIVO + IPI demonstrated significant activity in biomarker selected, pre-treated pts with mCRPC. The safety profile is consistent with this dosing schedule. Accrual to expansion cohort 2 started in September 2020. Determinants of immunogenic signature in responding patientsPatientInflammatory Infiltrate (%)MMRD (Y/N)DDRD (Y/N)TMB Mut/MBNEP-03340NN1NEP-03610NY Biallelic BRCA24NEP-0964NY Biallelic BRCA28NEP-1093Y MSH2/MSH6N14NEP-12625Y MSH2/MSH6N21NEP-13110Y PMS2/MLH1N13NEP-15920Y MSH2/MSH6Y BRCA127NEP-16525NY Biallelic CDK125NEP-06930NN1 Citation Format: Mark D. Linch, Yien Ning Sophia Wong, Robert Jones, Peter Sankey, Debra H. Josephs, Simon J. Crabb, John Staffurth, Anjali Zarkar, Laura White, Marian Duggan, Giulia Pellizzari, Graham Wheeler, Sandy Beare, Hayley Cartwright, Josep Linares, Ayse Akarca, Sergio A. Quezada, Leah Ensell, John Hartley, Gerhardt Attard, Joanna Burr, Anuradha Jayaram, Bihani Kularatne, Mahaz Kayani, Moon Chung, Colin C. Pritchard, Alex Freeman, Aiman Haider, Teresa Marafioti. Nivolumab (NIVO) and ipilimumab (IPI) treatment in prostate cancer with an immunogenic signature: cohort 1 of the NEPTUNES multi-centre, two-stage biomarker-selected Phase II trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB004.

To the editor: The central nervous system (CNS) is a key site of extramedullary disease in pediatric acute lymphoblastic leukemia (ALL),[1][1] and prior to the development of contemporary risk-adapted treatment strategies, CNS involvement was inevitable in most cases.[1][1] However, the biology of

We investigated and modeled the mesenchymal stromal cell (MSC) niche in adult acute lymphoblastic leukemia (ALL). We used gene expression profiling, cytokine/chemokine quantification, flow cytometry, and a variety of imaging techniques to show that MSCs, directly isolated from the primary bone marrow specimens of patients with ALL, frequently adopted an activated, cancer-associated fibroblast phenotype. Normal, primary human MSCs and the MSC cell line HS27a both were activated de novo, when exposed to the reactive oxygen species (ROS)–inducing chemotherapy agents cytarabine (AraC) and daunorubicin (DNR), a phenomenon blocked by the antioxidant N-acetyl cysteine. Chemotherapy-activated HS27a cells were functionally evaluated in a coculture model with ALL targets. Activated MSCs prevented therapy-induced apoptosis and death in ALL targets, via mitochondrial transfer through tunneling nanotubes (TNTs). Reduction of mitochondrial transfer by selective mitochondrial depletion or interference with TNT formation by microtubule inhibitors, such as vincristine (VCR), prevented the “rescue” function of activated MSCs. Corticosteroids, also a mainstay of ALL therapy, prevented the activation of MSCs. We also demonstrated that AraC (but not VCR) induced activation of MSCs, mitochondrial transfer, and mitochondrial mass increase in a murine NSG model of disseminated SEM cell–derived ALL, wherein CD19(+) cells closely associated with nestin(+) MSCs after AraC, but not in the other conditions. Our data propose a readily clinically exploitable mechanism for improving treatment of ALL, in which traditional ROS-inducing chemotherapies are often ineffective at eradicating residual disease, despite efficiently killing the bulk population.