Proteogenomic characterization of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor patient survival. Toward understanding the underlying molecular alterations that drive PDAC oncogenesis, we conducted comprehensive proteogenomic analysis of 140 pancreatic cancers, 67 normal adjacent tissues, and 9 normal pancreatic ductal tissues. Proteomic, phosphoproteomic, and glycoproteomic analyses were used to characterize proteins and their modifications. In addition, whole-genome sequencing, whole-exome sequencing, methylation, RNA sequencing (RNA-seq), and microRNA sequencing (miRNA-seq) were performed on the same tissues to facilitate an integrated proteogenomic analysis and determine the impact of genomic alterations on protein expression, signaling pathways, and post-translational modifications. To ensure robust downstream analyses, tumor neoplastic cellularity was assessed via multiple orthogonal strategies using molecular features and verified via pathological estimation of tumor cellularity based on histological review. This integrated proteogenomic characterization of PDAC will serve as a valuable resource for the community, paving the way for early detection and identification of novel therapeutic targets.

Profiling of syngeneic mouse HCC tumor models as a framework to understand anti-PD-1 sensitive tumor microenvironments.

BACKGROUND AND AIMS: The treatment of hepatocellular carcinoma (HCC) has been transformed by the use of immune checkpoint inhibitors. However, most patients with HCC do not benefit from treatment with immunotherapy. There is an urgent need to understand the mechanisms that underlie response or resistance to immunotherapy for patients with HCC. The use of syngeneic mouse models that closely recapitulate the heterogeneity of human HCC will provide opportunities to examine the complex interactions between cancer cells and nonmalignant cells in the tumor microenvironment. APPROACH AND RESULTS: We leverage a multifaceted approach that includes imaging mass cytometry and suspension cytometry by time of flight to profile the tumor microenvironments of the Hep53.4, Hepa 1-6, RIL-175, and TIBx (derivative of TIB-75) syngeneic mouse HCC models. The immune tumor microenvironments vary across these four models, and various immunosuppressive pathways exist at baseline in orthotopic liver tumors derived from these models. For instance, TIBx, which is resistant to anti-programmed cell death protein 1 therapy, contains a high proportion of "M2-like" tumor-associated macrophages with the potential to diminish antitumor immunity. Investigation of The Cancer Genome Atlas reveals that the baseline immunologic profiles of Hep53.4, RIL-175, and TIBx are broadly representative of human HCCs; however, Hepa 1-6 does not recapitulate the immune tumor microenvironment of the vast majority of human HCCs. CONCLUSIONS: There is a wide diversity in the immune tumor microenvironments in preclinical models and in human HCC, highlighting the need to use multiple syngeneic HCC models to improve the understanding of how to treat HCC through immune modulation.

Revisiting the tumorigenesis timeline with a data-driven generative model.

Cancer is driven by the sequential accumulation of genetic and epigenetic changes in oncogenes and tumor suppressor genes. The timing of these events is not well understood. Moreover, it is currently unknown why the same driver gene change appears as an early event in some cancer types and as a later event, or not at all, in others. These questions have become even more topical with the recent progress brought by genome-wide sequencing studies of cancer. Focusing on mutational events, we provide a mathematical model of the full process of tumor evolution that includes different types of fitness advantages for driver genes and carrying-capacity considerations. The model is able to recapitulate a substantial proportion of the observed cancer incidence in several cancer types (colorectal, pancreatic, and leukemia) and inherited conditions (Lynch and familial adenomatous polyposis), by changing only 2 tissue-specific parameters: the number of stem cells in a tissue and its cell division frequency. The model sheds light on the evolutionary dynamics of cancer by suggesting a generalized early onset of tumorigenesis followed by slow mutational waves, in contrast to previous conclusions. Formulas and estimates are provided for the fitness increases induced by driver mutations, often much larger than previously described, and highly tissue dependent. Our results suggest a mechanistic explanation for why the selective fitness advantage introduced by specific driver genes is tissue dependent.

Novel Genes Involved in Hypertrophic Cardiomyopathy: Data of Transcriptome and Methylome Profiling.

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease; its pathogenesis is still being intensively studied to explain the reasons for the significant genetic and phenotypic heterogeneity of the disease. To search for new genes involved in HCM development, we analyzed gene expression profiles coupled with DNA methylation profiles in the hypertrophied myocardia of HCM patients. The transcriptome analysis identified significant differences in the levels of 193 genes, most of which were underexpressed in HCM. The methylome analysis revealed 1755 nominally significant differentially methylated positions (DMPs), mostly hypomethylated in HCM. Based on gene ontology enrichment analysis, the majority of biological processes, overrepresented by both differentially expressed genes (DEGs) and DMP-containing genes, are involved in the regulation of locomotion and muscle structure development. The intersection of 193 DEGs and 978 DMP-containing genes pinpointed eight common genes, the expressions of which correlated with the methylation levels of the neighboring DMPs. Half of these genes (AUTS2, BRSK2, PRRT1, and SLC17A7), regulated by the mechanism of DNA methylation, were underexpressed in HCM and were involved in neurogenesis and synapse functioning. Our data, suggesting the involvement of innervation-associated genes in HCM, provide additional insights into disease pathogenesis and expand the field of further research.

Diagnostic utility of telomere length testing in a hospital-based setting.

Telomere length (TL) predicts the onset of cellular senescence in vitro but the diagnostic utility of TL measurement in clinical settings is not fully known. We tested the value of TL measurement by flow cytometry and FISH (flowFISH) in patients with mutations in telomerase and telomere maintenance genes. TL had a discrete and reproducible normal range with definable upper and lower boundaries. While TL above the 50th age-adjusted percentile had a 100% negative predictive value for clinically relevant mutations, the lower threshold in mutation carriers was age-dependent, and adult mutation carriers often overlapped with the lowest decile of controls. The extent of telomere shortening correlated with the age at diagnosis as well as the short telomere syndrome phenotype. Extremely short TL caused bone marrow failure and immunodeficiency in children and young adults, while milder defects manifested as pulmonary fibrosis-emphysema in adults. We prospectively examined whether TL altered treatment decisions for newly diagnosed idiopathic bone marrow failure patients and found abnormally short TL enriched for patients with mutations in some inherited bone marrow failure genes, such as RUNX1, in addition to telomerase and telomere maintenance genes. The result was actionable, altering the choice of treatment regimen and/or hematopoietic stem cell donor in one-fourth of the cases (9 of 38, 24%). We conclude that TL measurement by flowFISH, when used for targeted clinical indications and in limited settings, can influence treatment decisions in ways that improve outcome.

Inferring causal molecular networks: empirical assessment through a community-based effort.

It remains unclear whether causal, rather than merely correlational, relationships in molecular networks can be inferred in complex biological settings. Here we describe the HPN-DREAM network inference challenge, which focused on learning causal influences in signaling networks. We used phosphoprotein data from cancer cell lines as well as in silico data from a nonlinear dynamical model. Using the phosphoprotein data, we scored more than 2,000 networks submitted by challenge participants. The networks spanned 32 biological contexts and were scored in terms of causal validity with respect to unseen interventional data. A number of approaches were effective, and incorporating known biology was generally advantageous. Additional sub-challenges considered time-course prediction and visualization. Our results suggest that learning causal relationships may be feasible in complex settings such as disease states. Furthermore, our scoring approach provides a practical way to empirically assess inferred molecular networks in a causal sense.

Association of PD-1/PD-L axis expression with cytolytic activity, mutational load, and prognosis in melanoma and other solid tumors.

Programmed cell death protein-1 (PD-1)/programmed death ligand-1 (PD-L1) checkpoint blockade has led to remarkable and durable objective responses in a number of different tumor types. A better understanding of factors associated with the PD-1/PD-L axis expression is desirable, as it informs their potential role as prognostic and predictive biomarkers and may suggest rational treatment combinations. In the current study, we analyzed PD-L1, PD-L2, PD-1, and cytolytic activity (CYT) expression, as well as mutational density from melanoma and eight other solid tumor types using The Cancer Genome Atlas database. We found that in some tumor types, PD-L2 expression is more closely linked to Th1/IFNG expression and PD-1 and CD8 signaling than PD-L1 In contrast, mutational load was not correlated with a Th1/IFNG gene signature in any tumor type. PD-L1, PD-L2, PD-1, CYT expression, and mutational density are all positive prognostic features in melanoma, and conditional inference modeling revealed PD-1/CYT expression (i.e., an inflamed tumor microenvironment) as the most impactful feature, followed by mutational density. This study elucidates the highly interdependent nature of these parameters, and also indicates that future biomarkers for anti-PD-1/PD-L1 will benefit from tumor-type-specific, integrated, mRNA, protein, and genomic approaches.

Discovery and development of differentially methylated regions in human papillomavirus-related oropharyngeal squamous cell carcinoma.

Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) exhibits a different composition of epigenetic alterations. In this study, we identified differentially methylated regions (DMRs) with potential utility in screening for HPV-positive OPSCC. Genome wide DNA methylation was measured using methyl-CpG binding domain protein-enriched genome sequencing (MBD-seq) in 50 HPV-positive OPSCC tissues and 25 normal tissues. Fifty-one DMRs were defined with maximal methylation specificity to cancer samples. The Cancer Genome Atlas (TCGA) methylation array data was used to evaluate the performance of the proposed candidates. Supervised hierarchical clustering of 51 DMRs found that HPV-positive OPSCC had significantly higher DNA methylation levels compared to normal samples, and non-HPV-related head and neck squamous cell carcinoma (HNSCC). The methylation levels of all top 20 DNA methylation biomarkers in HPV-positive OPSCC were significantly higher than those in normal samples. Further confirmation using quantitative methylation specific PCR (QMSP) in an independent set of 24 HPV-related OPSCCs and 22 controls showed that 16 of the 20 candidates had significant higher methylation levels in HPV-positive OPSCC samples compared with controls. One candidate, OR6S1, had a sensitivity of 100%, while 17 candidates (KCNA3, EMBP1, CCDC181, DPP4, ITGA4, BEND4, ELMO1, SFMBT2, C1QL3, MIR129-2, NID2, HOXB4, ZNF439, ZNF93, VSTM2B, ZNF137P and ZNF773) had specificities of 100%. The prediction accuracy of the 20 candidates rang from 56.2% to 99.8% by receiver operating characteristic analysis. We have defined 20 highly specific DMRs in HPV-related OPSCC, which can potentially be applied to molecular-based detection tests and improve disease management.

Characterization of HPV and host genome interactions in primary head and neck cancers.

Previous studies have established that a subset of head and neck tumors contains human papillomavirus (HPV) sequences and that HPV-driven head and neck cancers display distinct biological and clinical features. HPV is known to drive cancer by the actions of the E6 and E7 oncoproteins, but the molecular architecture of HPV infection and its interaction with the host genome in head and neck cancers have not been comprehensively described. We profiled a cohort of 279 head and neck cancers with next generation RNA and DNA sequencing and show that 35 (12.5%) tumors displayed evidence of high-risk HPV types 16, 33, or 35. Twenty-five cases had integration of the viral genome into one or more locations in the human genome with statistical enrichment for genic regions. Integrations had a marked impact on the human genome and were associated with alterations in DNA copy number, mRNA transcript abundance and splicing, and both inter- and intrachromosomal rearrangements. Many of these events involved genes with documented roles in cancer. Cancers with integrated vs. nonintegrated HPV displayed different patterns of DNA methylation and both human and viral gene expressions. Together, these data provide insight into the mechanisms by which HPV interacts with the human genome beyond expression of viral oncoproteins and suggest that specific integration events are an integral component of viral oncogenesis.

CHFR silencing or microsatellite instability is associated with increased antitumor activity of docetaxel or gemcitabine in colorectal cancer.

Phenotypic differences among cancers with the same origin may be associated with chemotherapy response. CHFR silencing associated with DNA methylation has been suggested to be predictive of taxane sensitivity in diverse tumor types. However, the use of microsatellite instability (MSI:unstable-MSS:stable) as a predictive marker for therapeutic effect has had conflicting results. We examined these molecular alterations as predictors of chemotherapy sensitivity in colorectal cancer (CRC). Differential sensitivity to docetaxel and gemcitabine was compared to potential predictive biomarkers CHFR methylation and MSI status. Cell lines that were MSI-H/CHFR-methylated, MSS/CHFR-methylated and MSS/CHFR-unmethylated were assessed for in vivo sensitivity of CRC cell line xenografts to docetaxel and/or gemcitabine. We observed increased sensitivity in vitro to gemcitabine in cell lines with MSI and docetaxel in cell lines with CHFR inactivation via DNA methylation. In vivo treatment of human xenografts confirmed differential sensitivity, with the MSI-H/CHFR-methylated line RKO having tumor growth inhibition to each agent, and at least additive tumor growth inhibition with combination therapy. The MSS-CHFR-unmethylated line, CACO2 , was resistant to single and combination therapy, while COLO205, the MSS/CHFR-methylated line, showed tumor growth inhibition with docetaxel, but not gemcitabine, therapy. CHFR methylation in CRC cell lines predicted for sensitivity in vitro and in vivo to docetaxel, while MSI-H cell lines were more sensitive to gemcitabine. These data suggest that a subset of CRC patients would be selectively sensitive to a novel combination of gemcitabine and docetaxel, and are the basis for an ongoing clinical trial of this combination in a biomarker-selected patient population.

MLL1 regulates cytokine-driven cell migration and metastasis.

Cell migration is a critical contributor to metastasis. Cytokine production and its role in cancer cell migration have been traditionally associated with immune cells. We find that the histone methyltransferase Mixed-Lineage Leukemia 1 (MLL1) controls 3D cell migration via cytokines, IL-6, IL-8, and TGF-ß1, secreted by the cancer cells themselves. MLL1, with its scaffold protein Menin, controls actin filament assembly via the IL-6/8/pSTAT3/Arp3 axis and myosin contractility via the TGF-ß1/Gli2/ROCK1/2/pMLC2 axis, which together regulate dynamic protrusion generation and 3D cell migration. MLL1 also regulates cell proliferation via mitosis-based and cell cycle-related pathways. Mice bearing orthotopic MLL1-depleted tumors exhibit decreased lung metastatic burden and longer survival. MLL1 depletion leads to lower metastatic burden even when controlling for the difference in primary tumor growth rates. Combining MLL1-Menin inhibitor with paclitaxel abrogates tumor growth and metastasis, including preexistent metastasis. These results establish MLL1 as a potent regulator of cell migration and highlight the potential of targeting MLL1 in patients with metastatic disease.

Association between pathologic response and survival after neoadjuvant therapy in lung cancer.

Neoadjuvant immunotherapy plus chemotherapy improves event-free survival (EFS) and pathologic complete response (0% residual viable tumor (RVT) in primary tumor (PT) and lymph nodes (LNs)), and is approved for treatment of resectable lung cancer. Pathologic response assessment after neoadjuvant therapy is the potential analog to radiographic response for advanced disease. However, %RVT thresholds beyond pathologic complete response and major pathologic response (≤10% RVT) have not been explored. Pathologic response was prospectively assessed in the randomized, phase 3 CheckMate 816 trial (NCT02998528), which evaluated neoadjuvant nivolumab (anti-programmed death protein 1) plus chemotherapy in patients with resectable lung cancer. RVT, regression and necrosis were quantified (0-100%) in PT and LNs using a pan-tumor scoring system and tested for association with EFS in a prespecified exploratory analysis. Regardless of LN involvement, EFS improved with 0% versus >0% RVT-PT (hazard ratio = 0.18). RVT-PT predicted EFS for nivolumab plus chemotherapy (area under the curve = 0.74); 2-year EFS rates were 90%, 60%, 57% and 39% for patients with 0-5%, >5-30%, >30-80% and >80% RVT, respectively. Each 1% RVT associated with a 0.017 hazard ratio increase for EFS. Combining pathologic response from PT and LNs helped differentiate outcomes. When compared with radiographic response and circulating tumor DNA clearance, %RVT best approximated EFS. These findings support pathologic response as an emerging survival surrogate. Further assessment of the full spectrum of %RVT in lung cancer and other tumor types is warranted. ClinicalTrials.gov registration: NCT02998528 .

Entinostat, nivolumab and ipilimumab for women with advanced HER2-negative breast cancer: a phase Ib trial.

We report the results of 24 women, 50% (N = 12) with hormone receptor-positive breast cancer and 50% (N = 12) with advanced triple-negative breast cancer, treated with entinostat + nivolumab + ipilimumab from the dose escalation (N = 6) and expansion cohort (N = 18) of ETCTN-9844 ( NCT02453620 ). The primary endpoint was safety. Secondary endpoints were overall response rate, clinical benefit rate, progression-free survival and change in tumor CD8:FoxP3 ratio. There were no dose-limiting toxicities. Among evaluable participants (N = 20), the overall response rate was 25% (N = 5), with 40% (N = 4) in triple-negative breast cancer and 10% (N = 1) in hormone receptor-positive breast cancer. The clinical benefit rate was 40% (N = 8), and progression-free survival at 6 months was 50%. Exploratory analyses revealed that changes in myeloid cells may contribute to responses; however, no correlation was noted between changes in CD8:FoxP3 ratio, PD-L1 status and tumor mutational burden and response. These findings support further investigation of this treatment in a phase II trial.

Transfer Learning Reveals Cancer-Associated Fibroblasts Are Associated with Epithelial-Mesenchymal Transition and Inflammation in Cancer Cells in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by an immunosuppressive tumor microenvironment enriched with cancer-associated fibroblasts (CAF). This study used a convergence approach to identify tumor cell and CAF interactions through the integration of single-cell data from human tumors with human organoid coculture experiments. Analysis of a comprehensive atlas of PDAC single-cell RNA sequencing data indicated that CAF density is associated with increased inflammation and epithelial-mesenchymal transition (EMT) in epithelial cells. Transfer learning using transcriptional data from patient-derived organoid and CAF cocultures provided in silico validation of CAF induction of inflammatory and EMT epithelial cell states. Further experimental validation in cocultures demonstrated integrin beta 1 (ITGB1) and vascular endothelial factor A (VEGFA) interactions with neuropilin-1 mediating CAF-epithelial cell cross-talk. Together, this study introduces transfer learning from human single-cell data to organoid coculture analyses for experimental validation of discoveries of cell-cell cross-talk and identifies fibroblast-mediated regulation of EMT and inflammation. SIGNIFICANCE: Adaptation of transfer learning to relate human single-cell RNA sequencing data to organoid-CAF cocultures facilitates discovery of human pancreatic cancer intercellular interactions and uncovers cross-talk between CAFs and tumor cells through VEGFA and ITGB1.

Spatial transcriptomics analysis of neoadjuvant cabozantinib and nivolumab in advanced hepatocellular carcinoma identifies independent mechanisms of resistance and recurrence.

Novel immunotherapy combination therapies have improved outcomes for patients with hepatocellular carcinoma (HCC), but responses are limited to a subset of patients and recurrence can also occur. Little is known about the inter- and intra-tumor heterogeneity in cellular signaling networks within the HCC tumor microenvironment (TME) that underlie responses to modern systemic therapy. We applied spatial transcriptomics (ST) profiling to characterize the tumor microenvironment in HCC resection specimens from a clinical trial of neoadjuvant cabozantinib, a multi-tyrosine kinase inhibitor that primarily blocks VEGF, and nivolumab, a PD-1 inhibitor in which 5 out of 15 patients were found to have a pathologic response. ST profiling demonstrated that the TME of responding tumors was enriched for immune cells and cancer associated fibroblasts (CAF) with pro-inflammatory signaling relative to the non-responders. The enriched cancer-immune interactions in responding tumors are characterized by activation of the PAX5 module, a known regulator of B cell maturation, which colocalized with spots with increased B cell markers expression suggesting strong activity of these cells. Cancer-CAF interactions were also enriched in the responding tumors and were associated with extracellular matrix (ECM) remodeling as there was high activation of FOS and JUN in CAFs adjacent to tumor. The ECM remodeling is consistent with proliferative fibrosis in association with immune-mediated tumor regression. Among the patients with major pathologic response, a single patient experienced early HCC recurrence. ST analysis of this clinical outlier demonstrated marked tumor heterogeneity, with a distinctive immune-poor tumor region that resembles the non-responding TME across patients and was characterized by cancer-CAF interactions and expression of cancer stem cell markers, potentially mediating early tumor immune escape and recurrence in this patient. These data show that responses to modern systemic therapy in HCC are associated with distinctive molecular and cellular landscapes and provide new targets to enhance and prolong responses to systemic therapy in HCC.

Combinatorial biomarker for predicting outcomes to anti-PD-1 therapy in patients with metastatic clear cell renal cell carcinoma.

With a rapidly developing immunotherapeutic landscape for patients with metastatic clear cell renal cell carcinoma, biomarkers of efficacy are highly desirable to guide treatment strategy. Hematoxylin and eosin (H&E)-stained slides are inexpensive and widely available in pathology laboratories, including in resource-poor settings. Here, H&E scoring of tumor-infiltrating immune cells (TILplus) in pre-treatment tumor specimens using light microscopy is associated with improved overall survival (OS) in three independent cohorts of patients receiving immune checkpoint blockade. Necrosis score alone does not associate with OS; however, necrosis modifies the predictive effect of TILplus, a finding that has broad translational relevance for tissue-based biomarker development. PBRM1 mutational status is combined with H&E scores to further refine outcome predictions (OS, p = 0.007, and objective response, p = 0.04). These findings bring H&E assessment to the fore for biomarker development in future prospective, randomized trials, and emerging multi-omics classifiers.

Spatial transcriptomics analysis of neoadjuvant cabozantinib and nivolumab in advanced hepatocellular carcinoma identifies independent mechanisms of resistance and recurrence.

BACKGROUND: Novel immunotherapy combination therapies have improved outcomes for patients with hepatocellular carcinoma (HCC), but responses are limited to a subset of patients. Little is known about the inter- and intra-tumor heterogeneity in cellular signaling networks within the HCC tumor microenvironment (TME) that underlie responses to modern systemic therapy. METHODS: We applied spatial transcriptomics (ST) profiling to characterize the tumor microenvironment in HCC resection specimens from a prospective clinical trial of neoadjuvant cabozantinib, a multi-tyrosine kinase inhibitor that primarily blocks VEGF, and nivolumab, a PD-1 inhibitor in which 5 out of 15 patients were found to have a pathologic response at the time of resection. RESULTS: ST profiling demonstrated that the TME of responding tumors was enriched for immune cells and cancer-associated fibroblasts (CAF) with pro-inflammatory signaling relative to the non-responders. The enriched cancer-immune interactions in responding tumors are characterized by activation of the PAX5 module, a known regulator of B cell maturation, which colocalized with spots with increased B cell marker expression suggesting strong activity of these cells. HCC-CAF interactions were also enriched in the responding tumors and were associated with extracellular matrix (ECM) remodeling as there was high activation of FOS and JUN in CAFs adjacent to the tumor. The ECM remodeling is consistent with proliferative fibrosis in association with immune-mediated tumor regression. Among the patients with major pathologic responses, a single patient experienced early HCC recurrence. ST analysis of this clinical outlier demonstrated marked tumor heterogeneity, with a distinctive immune-poor tumor region that resembles the non-responding TME across patients and was characterized by HCC-CAF interactions and expression of cancer stem cell markers, potentially mediating early tumor immune escape and recurrence in this patient. CONCLUSIONS: These data show that responses to modern systemic therapy in HCC are associated with distinctive molecular and cellular landscapes and provide new targets to enhance and prolong responses to systemic therapy in HCC.

Myo1e overexpression in lung adenocarcinoma is associated with increased risk of mortality.

This study aims to perform a comprehensive genomic analysis to assess the influence of overexpression of MYO1E in non-small cell lung carcinoma (NSCLC) and whether there are differences in survival and mortality risk in NSCLC patients depending on both DNA methylation and RNA expression of MYO1E. The DNA methylation probe cg13887966 was inversely correlated with MYO1E RNA expression in both LUAD and LUSC subpopulations showing that lower MYO1E RNA expression was associated with higher MYO1E DNA methylation. Late stages of lung cancer showed significantly lower MYO1E DNA methylation and significantly higher MYO1E RNA expression for LUAD but not for LUSC. Low DNA methylation as well as high RNA expression of MYO1E are associated with a shorter median survival time and an increased risk of mortality for LUAD, but not for LUSC. This study suggests that changes in MYO1E methylation and expression in LUAD patients may have an essential role in lung cancer's pathogenesis. It shows the utility of MYO1E DNA methylation and RNA expression in predicting survival for LUAD patients. Also, given the low normal expression of MYO1E in blood cells MYO1E DNA methylation has the potential to be used as circulating tumor marker in liquid biopsies.

Uncovering the spatial landscape of molecular interactions within the tumor microenvironment through latent spaces.

Recent advances in spatial transcriptomics (STs) enable gene expression measurements from a tissue sample while retaining its spatial context. This technology enables unprecedented in situ resolution of the regulatory pathways that underlie the heterogeneity in the tumor as well as the tumor microenvironment (TME). The direct characterization of cellular co-localization with spatial technologies facilities quantification of the molecular changes resulting from direct cell-cell interaction, as it occurs in tumor-immune interactions. We present SpaceMarkers, a bioinformatics algorithm to infer molecular changes from cell-cell interactions from latent space analysis of ST data. We apply this approach to infer the molecular changes from tumor-immune interactions in Visium spatial transcriptomics data of metastasis, invasive and precursor lesions, and immunotherapy treatment. Further transfer learning in matched scRNA-seq data enabled further quantification of the specific cell types in which SpaceMarkers are enriched. Altogether, SpaceMarkers can identify the location and context-specific molecular interactions within the TME from ST data.

Immune landscape of tertiary lymphoid structures in hepatocellular carcinoma (HCC) treated with neoadjuvant immune checkpoint blockade.

Neoadjuvant immunotherapy is thought to produce long-term remissions through induction of antitumor immune responses before removal of the primary tumor. Tertiary lymphoid structures (TLS), germinal center-like structures that can arise within tumors, may contribute to the establishment of immunological memory in this setting, but understanding of their role remains limited. Here, we investigated the contribution of TLS to antitumor immunity in hepatocellular carcinoma (HCC) treated with neoadjuvant immunotherapy. We found that neoadjuvant immunotherapy induced the formation of TLS, which were associated with superior pathologic response, improved relapse free survival, and expansion of the intratumoral T and B cell repertoire. While TLS in viable tumor displayed a highly active mature morphology, in areas of tumor regression we identified an involuted TLS morphology, which was characterized by dispersion of the B cell follicle and persistence of a T cell zone enriched for ongoing antigen presentation and T cell-mature dendritic cell interactions. Involuted TLS showed increased expression of T cell memory markers and expansion of CD8+ cytotoxic and tissue resident memory clonotypes. Collectively, these data reveal the circumstances of TLS dissolution and suggest a functional role for late-stage TLS as sites of T cell memory formation after elimination of viable tumor.