AMBRA1 levels predict resistance to MAPK inhibitors in melanoma.

Intrinsic and acquired resistance to mitogen-activated protein kinase inhibitors (MAPKi) in melanoma remains a major therapeutic challenge. Here, we show that the clinical development of resistance to MAPKi is associated with reduced tumor expression of the melanoma suppressor Autophagy and Beclin 1 Regulator 1 (AMBRA1) and that lower expression levels of AMBRA1 predict a poor response to MAPKi treatment. Functional analyses show that loss of AMBRA1 induces phenotype switching and orchestrates an extracellular signal-regulated kinase (ERK)-independent resistance mechanism by activating focal adhesion kinase 1 (FAK1). In both in vitro and in vivo settings, melanomas with low AMBRA1 expression exhibit intrinsic resistance to MAPKi therapy but higher sensitivity to FAK1 inhibition. Finally, we show that the rapid development of resistance in initially MAPKi-sensitive melanomas can be attributed to preexisting subclones characterized by low AMBRA1 expression and that cotreatment with MAPKi and FAK1 inhibitors (FAKi) effectively prevents the development of resistance in these tumors. In summary, our findings underscore the value of AMBRA1 expression for predicting melanoma response to MAPKi and supporting the therapeutic efficacy of FAKi to overcome MAPKi-induced resistance.

Adoptive cell transfer therapy with ex vivo primed peripheral lymphocytes in combination with anti-PDL1 therapy effectively inhibits triple-negative breast cancer growth and metastasis.

BACKGROUND: Adoptive cell transfer cancer immunotherapy holds promise for treating disseminated disease, yet generating sufficient numbers of lymphocytes with anti-cancer activity against diverse specificities remains a major challenge. We recently developed a novel procedure (ALECSAT) for selecting, expanding and maturating polyclonal lymphocytes from peripheral blood with the capacity to target malignant cells. METHODS: Immunodeficient mice were challenged with triple-negative breast cancer cell lines or patient-derived xenografts (PDX) and treated with allogeneic or autologous ALECSAT cells with and without anti-PDL1 therapy to assess the capacity of ALECSAT cells to inhibit primary tumor growth and metastasis. RESULTS: ALECSAT mono therapy inhibited metastasis, but did not inhibit primary tumor growth or prolong survival of tumor-bearing mice. In contrast, combined ALECSAT and anti-PDL1 therapy significantly inhibited primary tumor growth, nearly completely blocked metastasis, and prolonged survival of tumor-bearing mice. CONCLUSIONS: Combined ALECSAT and anti-PDL1 therapy results in favorable anti-cancer responses in both cell line-derived xenograft and autologous PDX models of advanced triple-negative breast cancer.

Phenylalanine hydroxylase genotype-phenotype associations in the United States: A single center study.

Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism caused by pathogenic variants in the phenylalanine hydroxylase gene (PAH). The correlation between genotype and phenotype can be complex and sometimes variable but often very useful for categorizing and predicting dietary tolerance and potential outcome. We reviewed medical records for 367 patients diagnosed with PKU or persistent mild hyperphenylalaninemia (MHP) between 1950 and 2015 who had PAH genotyping. In 351 we had the full PAH genotype as well as phenotypic characteristics such as phenylalanine (Phe) concentrations (at newborn screening, confirmation, and highest known), and dietary Phe tolerance. On 716 mutant chromosomes, including 14 in genotypes with only one identified variant, we identified 114 different pathogenic variants. The most frequent, p.R408W, was present in 15.4% of the alleles; other frequent variants were c.1315 + 1G > A (6.1%), p.I65T (5.7%), and p.R261Q (5.7%). Three variants, c.142 T > G (p.L48 V), c.615G > C (p.E205D), and c.1342_1345delCTCC, were novel. We used the phenotypic parameters of variants paired with null alleles (functional hemizygotes) to assign the variants as classic PKU, moderate PKU, mild PKU, MHP-gray zone, or MHP. We also included the phenotype association(s) for all of the full genotypes. In 103 patients, we also could assign sapropterin dihydrochloride responsiveness, which is a synthetic form of the tetrahydrobiopterin (BH4) PAH cofactor. This compilation from a single metabolic center provides further information on PAH variants in the United States and the correlations between genotype and phenotype.

TP53 mutations identify younger mantle cell lymphoma patients who do not benefit from intensive chemoimmunotherapy.

Despite recent advances in lymphoma treatment, mantle cell lymphoma (MCL) remains incurable, and we are still unable to identify patients who will not benefit from the current standard of care. Here, we explore the prognostic value of recurrent genetic aberrations in diagnostic bone marrow (BM) specimens from 183 younger patients with MCL from the Nordic MCL2 and MCL3 trials, which represent current standard-of-care regimens. In the univariate model, mutations of TP53 (11%) and NOTCH1 (4%), and deletions of TP53 (16%) and CDKN2A (20%), were significantly associated with inferior outcomes (together with MIPI, MIPI-c, blastoid morphology, and Ki67 > 30%); however, in multivariate analyses, only TP53 mutations (HR, 6.2; P < .0001) retained prognostic impact for overall survival (OS), whereas TP53 mutations (HR, 6.9; P < .0001) and MIPI-c high-risk (HR, 2.6; P = .003) had independent prognostic impact on time to relapse. TP53-mutated cases had a dismal outcome, with a median OS of 1.8 years, and 50% relapsed at 1.0 years, compared to a median OS of 12.7 years for TP53-unmutated cases (P < .0001). TP53 mutations were significantly associated with Ki67 > 30%, blastoid morphology, MIPI high-risk, and inferior responses to both induction- and high-dose chemotherapy. In conclusion, we show that TP53 mutations identify a phenotypically distinct and highly aggressive form of MCL with poor or no response to regimens including cytarabine, rituximab, and autologous stem-cell transplant (ASCT). We suggest patients with MCL should be stratified according to TP53 status, and that patients with TP53 mutations should be considered for experimental frontline trials exploring novel agents.

DNA-Methylation-Based Detection of Urological Cancer in Urine: Overview of Biomarkers and Considerations on Biomarker Design, Source of DNA, and Detection Technologies.

Changes in DNA methylation have been causally linked with cancer and provide promising biomarkers for detection in biological fluids such as blood, urine, and saliva. The field has been fueled by genome-wide characterization of DNA methylation across cancer types as well as new technologies for sensitive detection of aberrantly methylated DNA molecules. For urological cancers, urine is in many situations the preferred "liquid biopsy" source because it contains exfoliated tumor cells and cell-free tumor DNA and can be obtained easily, noninvasively, and repeatedly. Here, we review recent advances made in the development of DNA-methylation-based biomarkers for detection of bladder, prostate, renal, and upper urinary tract cancers, with an emphasis on the performance characteristics of biomarkers in urine. For most biomarkers evaluated in independent studies, there was great variability in sensitivity and specificity. We discuss issues that impact the outcome of DNA-methylation-based detection of urological cancer and account for the great variability in performance, including genomic location of biomarkers, source of DNA, and technical issues related to the detection of rare aberrantly methylated DNA molecules. Finally, we discuss issues that remain to be addressed to fully exploit the potential of DNA-methylation-based biomarkers in the clinic, including the need for prospective trials and careful selection of control groups.

Noninvasive Detection of High Grade Prostate Cancer by DNA Methylation Analysis of Urine Cells Captured by Microfiltration.

PURPOSE: With the aim of developing a noninvasive test to detect clinically significant prostate cancer we investigated the potential of capturing cells from urine by microfiltration coupled with analysis of DNA methylation biomarkers. MATERIALS AND METHODS: In this prospective study urine from men suspected of having prostate cancer who were scheduled for transrectal ultrasound guided biopsy of the prostate was collected before digital rectal examination in 99, after digital rectal examination in 58 and from a urethral catheter in 7. Cells were isolated from whole volume voided urine using a filtration device containing a membrane filter with a pore size of 8 µm. Ten additional men provided 4 or 5 urine cell samples by self-collection prior to biopsy. Cellular DNA was analyzed for 5 methylation biomarkers using ddPCR™ (droplet digital polymerase chain reaction). RESULTS: Prostate cancer was diagnosed in 117 patients (71%). Across all tumor grades the sensitivity of urine DNA testing was 81% and 60% in samples obtained before and after digital rectal examination, respectively. In a prediction model including prostate specific antigen, patient age and the result of urine DNA analysis to detect high grade disease (Gleason score 7 or greater) an AUC of 0.95 (95% CI 0.90-1.00) was obtained for post-digital rectal examination samples. Analysis of repeat samples demonstrated substantial intraindividual variation in the shedding of cancerous cells in urine. CONCLUSIONS: Capturing cells from urine by microfiltration provides a novel tool to detect prostate cancer noninvasively with high sensitivity for high grade disease. Repeat sampling may increase sensitivity, particularly when urine is obtained without prior physical manipulation of the prostate.

Epigenetic remodelling and dysregulation of DLGAP4 is linked with early-onset cerebellar ataxia.

Genome instability, epigenetic remodelling and structural chromosomal rearrangements are hallmarks of cancer. However, the coordinated epigenetic effects of constitutional chromosomal rearrangements that disrupt genes associated with congenital neurodevelopmental diseases are poorly understood. To understand the genetic-epigenetic interplay at breakpoints of chromosomal translocations disrupting CG-rich loci, we quantified epigenetic modifications at DLGAP4 (SAPAP4), a key post-synaptic density 95 (PSD95) associated gene, truncated by the chromosome translocation t(8;20)(p12;q11.23), co-segregating with cerebellar ataxia in a five-generation family. We report significant epigenetic remodelling of the DLGAP4 locus triggered by the t(8;20)(p12;q11.23) translocation and leading to dysregulation of DLGAP4 expression in affected carriers. Disruption of DLGAP4 results in monoallelic hypermethylation of the truncated DLGAP4 promoter CpG island. This induced hypermethylation is maintained in somatic cells of carriers across several generations in a t(8;20) dependent-manner however, is erased in the germ cells of the translocation carriers. Subsequently, chromatin remodelling of the locus-perturbed monoallelic expression of DLGAP4 mRNAs and non-coding RNAs in haploid cells having the translocation. Our results provide new mechanistic insight into the way a balanced chromosomal rearrangement associated with a neurodevelopmental disorder perturbs allele-specific epigenetic mechanisms at breakpoints leading to the deregulation of the truncated locus.

Genome-wide DNA methylation analysis of transient neonatal diabetes type 1 patients with mutations in ZFP57.

BACKGROUND: Transient neonatal diabetes mellitus 1 (TNDM1) is a rare imprinting disorder characterized by intrautering growth retardation and diabetes mellitus usually presenting within the first six weeks of life and resolves by the age of 18 months. However, patients have an increased risk of developing diabetes mellitus type 2 later in life. Transient neonatal diabetes mellitus 1 is caused by overexpression of the maternally imprinted genes PLAGL1 and HYMAI on chromosome 6q24. One of the mechanisms leading to overexpression of the locus is hypomethylation of the maternal allele of PLAGL1 and HYMAI. A subset of patients with maternal hypomethylation at PLAGL1 have hypomethylation at additional imprinted loci throughout the genome, including GRB10, ZIM2 (PEG3), MEST (PEG1), KCNQ1OT1 and NESPAS (GNAS-AS1). About half of the TNDM1 patients carry mutations in ZFP57, a transcription factor involved in establishment and maintenance of methylation of imprinted loci. Our objective was to investigate whether additional regions are aberrantly methylated in ZFP57 mutation carriers. METHODS: Genome-wide DNA methylation analysis was performed on four individuals with homozygous or compound heterozygous ZFP57 mutations, three relatives with heterozygous ZFP57 mutations and five controls. Methylation status of selected regions showing aberrant methylation in the patients was verified using bisulfite-sequencing. RESULTS: We found large variability among the patients concerning the number and identity of the differentially methylated regions, but more than 60 regions were aberrantly methylated in two or more patients and a novel region within PPP1R13L was found to be hypomethylated in all the patients. The hypomethylated regions in common between the patients are enriched for the ZFP57 DNA binding motif. CONCLUSIONS: We have expanded the epimutational spectrum of TNDM1 associated with ZFP57 mutations and found one novel region within PPP1R13L which is hypomethylated in all TNDM1 patients included in this study. Functional studies of the locus might provide further insight into the etiology of the disease.

Mitochondrial DNA copy number and future risk of B-cell lymphoma in a nested case-control study in the prospective EPIC cohort.

It has been suggested that mitochondrial dysfunction and DNA damage are involved in lymphomagenesis. Increased copy number of mitochondrial DNA (mtDNA) as a compensatory mechanism of mitochondrial dysfunction previously has been associated with B-cell lymphomas, in particular chronic lymphocytic leukemia (CLL). However, current evidence is limited and based on a relatively small number of cases. Using a nested case-control study, we extended these findings with a focus on subtype-specific analyses. Relative mtDNA copy number was measured in the buffy coat of prospectively collected blood of 469 lymphoma cases and 469 matched controls. The association between mtDNA copy number and the risk of developing lymphoma and histologic subtypes was examined using logistic regression models. We found no overall association between mtDNA and risk of lymphoma. Subtype analyses revealed significant increased risks of CLL (n = 102) with increasing mtDNA copy number (odds ratio = 1.34, 1.44, and 1.80 for quartiles 2-4, respectively; P trend = .001). mtDNA copy number was not associated with follow-up time, suggesting that this observation is not strongly influenced by indolent disease status. This study substantially strengthens the evidence that mtDNA copy number is related to risk of CLL and supports the importance of mitochondrial dysfunction as a possible mechanistic pathway in CLL ontogenesis.

DNA methylation and histone modifications regulate SOX11 expression in lymphoid and solid cancer cells.

BACKGROUND: The neural transcription factor SOX11 is present at specific stages during embryo development with a very restricted expression in adult tissue, indicating precise regulation of transcription. SOX11 is strongly up-regulated in some malignancies and have a functional role in tumorgenesis. With the aim to explore differences in epigenetic regulation of SOX11 expression in normal versus neoplastic cells, we investigated methylation and histone modifications related to the SOX11 promoter and the possibility to induce re-expression using histone deacetylase (HDAC) or EZH2 inhibitors. METHODS: The epigenetic regulation of SOX11 was investigated in distinct non-malignant cell populations (n = 7) and neoplastic cell-lines (n = 42) of different cellular origins. DNA methylation was assessed using bisulfite sequencing, methylation-specific melting curve analysis, MethyLight and pyrosequencing. The presence of H3K27me3 was assessed using ChIP-qPCR. The HDAC inhibitors Vorinostat and trichostatin A were used to induce SOX11 in cell lines with no endogenous expression. RESULTS: The SOX11 promoter shows a low degree of methylation and strong enrichment of H3K27me3 in non-malignant differentiated cells, independent of cellular origin. Cancers of the B-cell lineage are strongly marked by de novo methylation at the SOX11 promoter in SOX11 non-expressing cells, while solid cancer entities display a more varying degree of SOX11 promoter methylation. The silencing mark H3K27me3 was generally present at the SOX11 promoter in non-expressing cells, and an increased enrichment was observed in cancer cells with a low degree of SOX11 methylation compared to cells with dense methylation. Finally, we demonstrate that the HDAC inhibitors (vorinostat and trichostatin A) induce SOX11 expression in cancer cells with low levels of SOX11 methylation. CONCLUSIONS: We show that SOX11 is strongly marked by repressive histone marks in non-malignant cells. In contrast, SOX11 regulation in neoplastic tissues is more complex involving both DNA methylation and histone modifications. The possibility to re-express SOX11 in non-methylated tissue is of clinical relevance, and was successfully achieved in cell lines with low levels of SOX11 methylation. In breast cancer patients, methylation of the SOX11 promoter was shown to correlate with estrogen receptor status, suggesting that SOX11 may be functionally re-expressed during treatment with HDAC inhibitors in specific patient subgroups.

S100A4-neutralizing antibody suppresses spontaneous tumor progression, pre-metastatic niche formation and alters T-cell polarization balance.

BACKGROUND: The tumor microenvironment plays a determinative role in stimulating tumor progression and metastasis. Notably, tumor-stroma signals affect the pattern of infiltrated immune cells and the profile of tumor-released cytokines. Among the known molecules that are engaged in stimulating the metastatic spread of tumor cells is the S100A4 protein. S100A4 is known as an inducer of inflammatory processes and has been shown to attract T-cells to the primary tumor and to the pre-metastatic niche. The present study aims to examine the immunomodulatory role of S100A4 in vivo and in vitro and assess the mode of action of 6B12, a S100A4 neutralizing antibody. METHODS: The therapeutic effect of the 6B12 antibody was evaluated in two different mouse models. First, in a model of spontaneous breast cancer we assessed the dynamics of tumor growth and metastasis. Second, in a model of metastatic niche formation we determined the expression of metastatic niche markers. The levels of cytokine expression were assessed using antibody as well as PCR arrays and the results confirmed by qRT-PCR and ELISA. T-cell phenotyping and in vitro differentiation analyses were performed by flow cytometry. RESULTS: We show that the S100A4 protein alters the expression of transcription factor and signal transduction pathway genes involved in the T-cell lineage differentiation. T-cells challenged with S100A4 demonstrated reduced proportion of Th1-polarized cells shifting the Th1/Th2 balance towards the Th2 pro-tumorigenic phenotype. The 6B12 antibody restored the Th1/Th2 balance. Furthermore, we provide evidence that the 6B12 antibody deploys its anti-metastatic effect, by suppressing the attraction of T-cells to the site of primary tumor and pre-metastatic niche. This was associated with delayed primary tumor growth, decreased vessel density and inhibition of metastases. CONCLUSION: The S100A4 blocking antibody (6B12) reduces tumor growth and metastasis in a model of spontaneous breast cancer. The 6B12 antibody treatment inhibits T cell accumulation at the primary and pre-metastatic tumor sites. The 6B12 antibody acts as an immunomodulatory agent and thus supports the view that the 6B12 antibody is a promising therapeutic candidate to fight cancer.

Prediagnostic telomere length and risk of B-cell lymphoma-Results from the EPIC cohort study.

Recent epidemiological investigations have reported on the association between telomere length (TL) and a number of malignancies, including B-cell lymphoma (BCL). The reported results for BCLs are however inconsistent. We carried out a nested case-control study to determine whether TL is associated with future risk of BCL. Using quantitative polymerase chain reaction, the relative TL (i.e. the ratio of telomere repeat copy number to single gene copy number) was measured in mononuclear cell DNA of prediagnostic peripheral blood samples of 464 lymphoma cases and 464 matched controls (median time between blood collection and diagnosis, 4.6 years). Conditional logistic regression was used to analyze the association between TL and the risk of developing lymphoma and histologic subtypes. TL was significantly longer in cases compared to controls (p = 0.01). Multivariable models showed a significantly increased risk of BCL [odds ratio (OR) = 1.66, 1.80 and 3.20 for quartiles 2-4, respectively, p-trend = 0.001], diffuse large B-cell lymphoma (DLBCL) (OR = 1.20, 2.48 and 2.36 for quartiles 2-4, respectively, p-trend = 0.03) and follicular lymphoma (FL) (OR = 1.39, 1.90 and 2.69 for quartiles 2-4, respectively, p-trend = 0.02) with increasing TL. This study suggests an association between longer leucocyte TL and increased risk of BCL which was most pronounced for DLBCL and FL.

Persistence of DNMT3A mutations at long-term remission in adult patients with AML.

Mutations in DNMT3A, the gene encoding DNA methyltransferase 3 alpha, have been identified as molecular drivers in acute myeloid leukaemia (AML) with possible implications for minimal residual disease monitoring and prognosis. To further explore the utility of DNMT3A mutations as biomarkers for AML, we developed assays for sensitive detection of recurrent mutations affecting residue R882. Analysis of DNA from 298 diagnostic AML samples revealed DNMT3A mutations in 45 cases (15%), which coincided with mutations in NPM1, FLT3 and IDH1. DNMT3A mutations were stable in 12 of 13 patients presenting with relapse or secondary myelodysplastic syndrome, but were also present in remission samples from 14 patients (at allele frequencies of <1-50%) up to 8 years after initial AML diagnosis, despite the loss of all other molecular AML markers. The mutant DNMT3A allele burden was not related to the clinical course of disease. Cell sorting demonstrated the presence of DNMT3A mutations in leukaemic blasts, but also at lower allele frequencies in T and B-cells from the same patients. Our data are consistent with the recent finding of preleukaemic stem cells in AML, which are resistant to chemotherapy. The persistence of DNMT3A mutations during remission may have important implications for the management of AML.

Bioenergetic modulation with dichloroacetate reduces the growth of melanoma cells and potentiates their response to BRAFV600E inhibition.

BACKGROUND: Advances in melanoma treatment through targeted inhibition of oncogenic BRAF are limited owing to the development of acquired resistance. The involvement of BRAFV600E in metabolic reprogramming of melanoma cells provides a rationale for co-targeting metabolism as a therapeutic approach. METHODS: We examined the effects of dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, on the growth and metabolic activity of human melanoma cell lines. The combined effect of DCA and the BRAF inhibitor vemurafenib was investigated in BRAFV600E -mutated melanoma cell lines. Vemurafenib-resistant cell lines were established in vitro and their sensitivity to DCA was tested. RESULTS: DCA induced a reduction in glycolytic activity and intracellular ATP levels, and inhibited cellular growth. Co-treatment of BRAFV600E-mutant melanoma cells with DCA and vemurafenib induced a greater reduction in intracellular ATP levels and cellular growth than either compound alone. In addition, melanoma cells with in vitro acquired resistance to vemurafenib retained their sensitivity to DCA. CONCLUSIONS: These results suggest that DCA potentiates the effect of vemurafenib through a cooperative attenuation of energy production. Furthermore, the demonstration of retained sensitivity to DCA in melanoma cells with acquired resistance to vemurafenib could have implications for melanoma treatment.

Age-related differences in cancer biology in older patients.

Aging impacts cancer development with incidence rising exponentially. Age-related genetic and epigenetic changes, along with the aging microenvironment, contribute to cancer development. In older individuals, tumours manifest a heightened mutational burden and distinct genetic changes which differ significantly from tumours observed in younger patients. The aging microenvironment accumulates senescent cells, altered matrix, and a dysregulated immune system. Age-related changes in the immune system fuel tumour development and treatment resistance. Understanding these processes is crucial for optimized treatment of older patients with cancer, as discussed in this review.

Distinct longitudinal patterns of urine tumor DNA in patients undergoing surveillance for bladder cancer.

Cystoscopy is the gold standard for surveillance of non-muscle invasive bladder cancer (NMIBC), but the procedure is invasive and has suboptimal accuracy. The aim of this study was to investigate the potential of analyzing urine samples for the presence of urine tumor DNA (utDNA) to replace cystoscopy for surveillance of bladder cancer recurrence. In this longitudinal, prospective, and observational study, 47 patients were followed for recurrence for 2 years, involving analysis of utDNA using the BladMetrix DNA methylation biomarker test at each cystoscopy. In total, utDNA was detected in 21/23 recurrences (91% sensitivity), including 5/5 T1, T2, and carcinoma in situ (CIS) tumors (100%) and 10/12 Ta tumors (83%), with < 1% false-negative test results. Importantly, utDNA analysis showed the potential to reduce the number of cystoscopies by 55%, benefitting 79% of the patients. Eleven of 23 recurrences (48%) were detected earlier with utDNA than with cystoscopy, and distinct patterns of residual utDNA post-surgery indicated minimal residual disease (MRD) or field effect in 6% and 15% of the patients, respectively. In conclusion, utDNA analysis shows high sensitivity to detect tumor recurrence, potential to reduce the number of cystoscopies, and promise to guide patient-specific surveillance regimens.

Single-cell sequencing unveils extensive intratumoral heterogeneity of cancer/testis antigen expression in melanoma and lung cancer.

Cancer/testis antigens (CTAs) are widely expressed in melanoma and lung cancer, emerging as promising targets for vaccination strategies and T-cell-based therapies in these malignancies. Despite recognizing the essential impact of intratumoral heterogeneity on clinical responses to immunotherapy, our understanding of intratumoral heterogeneity in CTA expression has remained limited. We employed single-cell mRNA sequencing to delineate the CTA expression profiles of cancer cells in clinically derived melanoma and lung cancer samples. Our findings reveal a high degree of intratumoral transcriptional heterogeneity in CTA expression. In melanoma, every cell expressed at least one CTA. However, most individual CTAs, including the widely used therapeutic targets NY-ESO-1 and MAGE, were confined to subpopulations of cells and were uncoordinated in their expression, resulting in mosaics of cancer cells with diverse CTA profiles. Coordinated expression was observed, however, mainly among highly structurally and evolutionarily related CTA genes. Importantly, a minor subset of CTAs, including PRAME and several members of the GAGE and MAGE-A families, were homogenously expressed in melanomas, highlighting their potential as therapeutic targets. Extensive heterogeneity in CTA expression was also observed in lung cancer. However, the frequency of CTA-positive cancer cells was notably lower and homogenously expressed CTAs were only identified in one of five tumors in this cancer type. Our findings underscore the need for careful CTA target selection in immunotherapy development and clinical testing and offer a rational framework for identifying the most promising candidates.

Blood Leukocyte AHRR Methylation and Risk of Non-smoking-associated Cancer: A Case-cohort Study of Non-Hodgkin Lymphoma.

Aryl-hydrocarbon receptor repressor (AHRR) hypomethylation in peripheral blood is tightly linked with tobacco smoking and lung cancer. Here, we investigated AHRR methylation in non-Hodgkin lymphoma (NHL), a non-smoking-associated cancer. In a case-cohort study within the population-based Danish Diet, Cancer and Health cohort, we measured AHRR (cg23576855) methylation in prediagnostic blood from 161 participants who developed NHL within 13.4 years of follow-up (median: 8.5 years), with a comparison group of 164 randomly chosen participants. We measured DNA-methylation levels using bisulfite pyrosequencing and estimated incidence rate ratios (IRR) using Cox proportional hazards models with adjustment for baseline age, sex, educational level, smoking status, body mass index, alcohol intake, physical activity, and diet score. Global DNA-methylation levels were assessed by long interspersed nucleotide element 1 (LINE-1) analysis. Overall, the IRR for AHRR hypomethylation (lowest vs. other quartiles) was 2.52 [95% confidence interval (CI), 1.24-5.15]. When stratified according to time between blood draw and diagnosis, low AHRR methylation levels were associated with a future diagnosis of NHL [IRR: 4.50 (95% CI, 1.62-12.50) at 0-<5 years, 7.04 (95% CI, 2.36-21.02) at 5-<10 years, and 0.56 (95% CI, 0.21-1.45) at ≥10 years]. There was no association between global DNA-methylation levels and risk of NHL. Our results show that AHRR hypomethylation in blood leukocytes is associated with a higher risk of NHL in a time-dependent manner, suggesting that it occurs as a response to tumor development. Significance: Our population-based study demonstrated that lower AHRR methylation levels in peripheral blood leukocytes were associated with an increased risk of NHL. This association was independent of tobacco smoking, sex, and lifestyle characteristics, but was highly dependent on time to diagnosis. These findings highlight the potential of AHRR methylation as a biomarker for NHL risk, effective up to 10 years after blood draw.

DNA methyltransferase inhibition promotes recruitment of myeloid-derived suppressor cells to the tumor microenvironment through induction of tumor cell-intrinsic interleukin-1.

DNA methyltransferase (DNMT) inhibitors are used for treatment of certain hematological malignancies and exert anti-cancer activity through diverse mechanisms, including reexpression of tumor suppressor genes and anti-viral responses triggered by expression of endogenous retroviruses. Despite advances in the pharmacokinetic properties of DNMT inhibitors, the efficacy of these drugs in solid cancers remains low. Here, we show in cell lines and clinical and experimental tumors across multiple cancer types that DNMT inhibition induces the expression of interleukin-1 (IL-1), a cytokine with proinflammatory and protumorigenic properties. Specifically, this tumor-intrinsic IL-1 expression modulates the chemokine landscape of tumors and leads to the recruitment of monocytic myeloid-derived suppressor cells to the tumor microenvironment, processes that can be blocked by IL-1 antagonists. Molecular analysis demonstrates complex patterns of IL-1 and interferon activation and crosstalk in response to DNMT inhibition, which depend on the integrity of IRF- and NF-κB-mediated antiviral pathways and may determine the outcome of DNMT-inhibitor treatment. Together, our results show that DNMT inhibitors may negatively affect the microenvironment of a large subset of tumors and suggest that co-treatment with IL-1 antagonists may be a favorable combination for these patients.