[Development and validation of predictive models for esophageal squamous cell carcinoma and its precancerous lesions using terminal motif analysis in circulating cell-free DNA].

Objectives: To develop and validate predictive models for esophageal squamous cell carcinoma (ESCC) using circulating cell-free DNA (cfDNA) terminal motif analysis. The goal was to improve the non-invasive detection of early-stage ESCC and its precancerous lesions. Methods: Between August 2021 and November 2022, we prospectively collected plasma samples from 448 individuals at the Department of Endoscopy, Cancer Hospital, Chinese Academy of Medical Sciences for cfDNA extraction, library construction, and sequencing. We analyzed 201 cases of ESCC, 46 high-grade intraepithelial neoplasia (HGIN), 46 low-grade intraepithelial neoplasia (LGIN), 176 benign esophageal lesions, and 29 healthy controls. Participants, including ESCC patients and control subjects, were randomly assigned to a training set (n=284) and a validation set (n=122). The training cohort underwent z-score normalization of cfDNA terminal motif matrices and a selection of distinctive features differentiated ESCC cases from controls. The random forest classifier, Motif-1 (M1), was then developed through principal component analysis, ten-fold cross-validation, and recursive feature elimination. M1's efficacy was then validated in the validation and precancerous lesion sets. Subsequently, individuals with precancerous lesions were included in the dataset and participants were randomly allocated to newly formed training (n=243), validation (n=105), and test (n=150) cohorts. Using the same procedure as M1, we trained the Motif-2 (M2) random forest model with the training cohort. The M2 model's accuracy was then confirmed in the validation cohort to establish the optimal threshold and further tested by performing validation in the test cohort. Results: We developed two cfDNA terminal motif-based predictive models for ESCC and associated precancerous conditions. The first model, M1, achieved a sensitivity of 90.0%, a specificity of 77.4%, and an area under the curve (AUC) of 0.884 in the validation cohort. For LGIN, HGIN, and T1aN0 stage ESCC, M1's sensitivities were 76.1%, 80.4%, and 91.2% respectively. Notably, the sensitivity for jointly predicting HGIN and T1aN0 ESCC reached 85.0%. Both the predictive accuracy and sensitivity increased in line with the cancer's progression (P＜0.001). The second model, M2, exhibited a sensitivity of 87.5%, a specificity of 77.4%, and an AUC of 0.857 in the test cohort. M2's sensitivities for detecting precancerous lesions and ESCC were 80.0% and 89.7%, respectively, and it showed a combined sensitivity of 89.4% for HGIN and T1aN0 stage ESCC. Conclusions: Two predictive models based on cfDNA terminal motif analysis for ESCC and its precancerous lesions are developed. They both show high sensitivity and specificity in identifying ESCC and its precancerous stages, indicating its potential for early ESCC detection.

NEMO/NF-κB signaling functions as a double-edged sword in PanIN formation versus progression to pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is marked by a dismal survival rate, lacking effective therapeutics due to its aggressive growth, late-stage diagnosis, and chemotherapy resistance. Despite debates on NF-κB targeting for PDAC treatment, no successful approach has emerged. METHODS: To elucidate the role of NF-κB, we ablated NF-κB essential modulator (NEMO), critical for conventional NF-κB signaling, in the pancreata of mice that develop precancerous lesions (KC mouse model). Secretagogue-induced pancreatitis by cerulein injections was utilized to promote inflammation and accelerate PDAC development. RESULTS: NEMO deletion reduced fibrosis and inflammation in young KC mice, resulting in fewer pancreatic intraepithelial neoplasias (PanINs) at later stages. Paradoxically, however, NEMO deletion accelerated the progression of these fewer PanINs to PDAC and reduced median lifespan. Further, analysis of tissue microarrays from human PDAC sections highlighted the correlation between reduced NEMO expression in neoplastic cells and poorer prognosis, supporting our observation in mice. Mechanistically, NEMO deletion impeded oncogene-induced senescence (OIS), which is normally active in low-grade PanINs. This blockage resulted in fewer senescence-associated secretory phenotype (SASP) factors, reducing inflammation. However, blocked OIS fostered replication stress and DNA damage accumulation which accelerated PanIN progression to PDAC. Finally, treatment with the DNA damage-inducing reagent etoposide resulted in elevated cell death in NEMO-ablated PDAC cells compared to their NEMO-competent counterparts, indicative of a synthetic lethality paradigm. CONCLUSIONS: NEMO exhibited both oncogenic and tumor-suppressive properties during PDAC development. Caution is suggested in therapeutic interventions targeting NF-κB, which may be detrimental during PanIN progression but beneficial post-PDAC development.

DCLK1 Expression and its Functional Significance in Intrahepatic Cholangiocarcinoma and Bil-IN Stage.

BACKGROUND/AIM: Although several studies in some neoplasms have reported correlation between the expression levels of Doublecortin-like kinase1(DCLK1) and carcinogenesis, its role in cholangiocarcinoma remains unknown. MATERIALS AND METHODS: DCLK1 expression in normal epithelium (NE), biliary intraepithelial neoplasia (BilIN)1∼3, and intrahepatic cholangiocarcinoma (ICC) were investigated immuno-histochemically. The molecular effects of DCLK1 were investigated by gene silencing using RNAi [DCLK1-tagrgeting (siDCLK1)]. The human ICC cell lines HuCCT1 and HuH28 were transfected with these siRNAs, and used for assays in the presence or absence of DCLK1 inhibitors. RESULTS: The positive ratio of DCLK1 expression in ICC was higher than that in NE, and equally distributed among BilIN1∼3 (NE: BilIN1: BilIN2: BilIN3: ICC=62%: 91%: 97%: 100%: 95%, p<0.05). In the wound healing assay, the migration of the siDCLK1-treated cells was significantly inhibited compared to the NT-treated cells (p<0.05). In the cell invasion assay, the invasion of the siDCLK1-treated cells was significantly inhibited compared to the NT-treated cells (p<0.05). In the presence of the DCLK1 inhibitor, cell proliferative capacity at 24 hours was decreased in a concentration-dependent manner. CONCLUSION: DCLK1 was highly expressed in the early stage of ICC carcinogenesis. Human ICC cell growth was suppressed in vitro by siRNA silencing of DCLK1 or after treatment with the DCLK1 inhibitor, indicating DCLK1 may be molecular target for ICC therapy.

Stromal Rigidity Stress Accelerates Pancreatic Intraepithelial Neoplasia Progression and Chromosomal Instability via Nuclear Protein Tyrosine Kinase 2 Localization.

Because the mechanotransduction by stromal stiffness stimulates the rupture and repair of the nuclear envelope in pancreatic progenitor cells, accumulated genomic aberrations are under selection in the tumor microenvironment. Analysis of cell growth, micronuclei, and phosphorylated Ser-139 residue of the histone variant H2AX (γH2AX) foci linked to mechanotransduction pressure in vivo during serial orthotopic passages of mouse KrasLSL-G12D/+;Trp53flox/flox;Pdx1-Cre (KPC) cancer cells in the tumor and in migrating through the size-restricted 3-µm micropores. To search for pancreatic cancer cell-of-origin, analysis of single-cell data sets revealed that the extracellular matrix shaped an alternate route of acinar-ductal transdifferentiation of acinar cells into topoisomerase II α (TOP2A)-overexpressing cancer cells and derived subclusters with copy number amplifications in MYC-PTK2 (protein tyrosine kinase 2) locus and PIK3CA. High-PTK2 expression is associated with 171 differentially methylated CpG loci, 319 differentially expressed genes, and poor overall survival in The Cancer Genome Atlas-Pancreatic Adenocarcinoma cohort. Abolished RGD-integrin signaling by disintegrin KG blocked the PTK2 phosphorylation, increased cancer apoptosis, decreased vav guanine nucleotide exchange factor 1 (VAV1) expression, and prolonged overall survival in the KPC mice. Reduction of α-smooth muscle actin deposition in the CD248 knockout KPC mice remodeled the tissue stroma and down-regulated TOP2A expression in the epithelium. In summary, stromal stiffness induced the onset of cancer cells-of-origin by ectopic TOP2A expression, and the genomic amplification of MYC-PTK2 locus via alternative transdifferentiation of pancreatic progenitor cells is the vulnerability useful for disintegrin KG treatment.

Morphologic and Molecular Heterogeneity of High-grade Serous Carcinoma Precursor Lesions.

Serous tubal intraepithelial carcinoma (STIC) is the fallopian tube precursor lesion for most cases of pelvic high-grade serous carcinoma (HGSC). To date, the morphologic, molecular, and clinical heterogeneity of STIC and a less atypical putative precursor lesion, termed serous tubal intraepithelial lesion, has not been well characterized. Better understanding of precursor heterogeneity could impact the clinical management of women with incidental STICs (without concurrent carcinoma) identified in cases of prophylactic or opportunistic salpingectomy. This study analyzed morphologic and molecular features of 171 STICs and 21 serous tubal intraepithelial lesions. We assessed their histologic features, Ki-67 and p53 staining patterns, and genome-wide DNA copy number alterations. We classified all precursor lesions into 2 morphologic subtypes, one with a flat surface (Flat) and the other characterized by budding, loosely adherent, or detached (BLAD) morphology. On the basis of pathology review by a panel of 8 gynecologic pathologists, we found 87 BLAD, 96 Flat, and 9 indeterminate lesions. As compared with Flat lesions, BLAD lesions were more frequently diagnostic of STIC ( P <0.0001) and were found concurrently with HGSC ( P <0.0001). BLAD morphology was also characterized by higher Ki-67 proliferation index ( P <0.0001), presence of epithelial stratification ( P <0.0001), and increased lymphocyte density ( P <0.0001). BLAD lesions also exhibited more frequent DNA copy number gain/amplification at the CCNE1 or CMYC loci canonical to HGSCs ( P <0.0001). Both BLAD morphology and STIC diagnoses are independent risk factors for an elevated Ki-67 proliferation index. No correlation was observed between BLAD and Flat lesions with respect to patient age, presence of germline BRCA1/2 mutation, or p53 staining pattern. These findings suggest that tubal precursor lesions are morphologically and molecularly heterogeneous, laying the foundation for further studies on the pathogenesis of HGSC initiation and identifying histologic features predictive of poor patient outcomes.

Targeting NUPR1-dependent stress granules formation to induce synthetic lethality in KrasG12D-driven tumors.

We find that NUPR1, a stress-associated intrinsically disordered protein, induced droplet formation via liquid-liquid phase separation (LLPS). NUPR1-driven LLPS was crucial for the creation of NUPR1-dependent stress granules (SGs) in pancreatic cancer cells since genetic or pharmacological inhibition by ZZW-115 of NUPR1 activity impeded SGs formation. The KrasG12D mutation induced oncogenic stress, NUPR1 overexpression, and promoted SGs development. Notably, enforced NUPR1 expression induced SGs formation independently of mutated KrasG12D. Mechanistically, KrasG12D expression strengthened sensitivity to NUPR1 inactivation, inducing cell death, activating caspase 3 and releasing LDH. Remarkably, ZZW-115-mediated SG-formation inhibition hampered the development of pancreatic intraepithelial neoplasia (PanINs) in Pdx1-cre;LSL-KrasG12D (KC) mice. ZZW-115-treatment of KC mice triggered caspase 3 activation, DNA fragmentation, and formation of the apoptotic bodies, leading to cell death, specifically in KrasG12D-expressing cells. We further demonstrated that, in developed PanINs, short-term ZZW-115 treatment prevented NUPR1-associated SGs presence. Lastly, a four-week ZZW-115 treatment significantly reduced the number and size of PanINs in KC mice. This study proposes that targeting NUPR1-dependent SGs formation could be a therapeutic approach to induce cell death in KrasG12D-dependent tumors.

Oral bacteria accelerate pancreatic cancer development in mice.

OBJECTIVE: Epidemiological studies highlight an association between pancreatic ductal adenocarcinoma (PDAC) and oral carriage of the anaerobic bacterium Porphyromonas gingivalis, a species highly linked to periodontal disease. We analysed the potential for P. gingivalis to promote pancreatic cancer development in an animal model and probed underlying mechanisms. DESIGN: We tracked P. gingivalis bacterial translocation from the oral cavity to the pancreas following administration to mice. To dissect the role of P. gingivalis in PDAC development, we administered bacteria to a genetically engineered mouse PDAC model consisting of inducible acinar cell expression of mutant Kras (Kras +/LSL-G12D; Ptf1a-CreER, iKC mice). These mice were used to study the cooperative effects of Kras mutation and P. gingivalis on the progression of pancreatic intraepithelial neoplasia (PanIN) to PDAC. The direct effects of P. gingivalis on acinar cells and PDAC cell lines were studied in vitro. RESULTS: P. gingivalis migrated from the oral cavity to the pancreas in mice and can be detected in human PanIN lesions. Repetitive P. gingivalis administration to wild-type mice induced pancreatic acinar-to-ductal metaplasia (ADM), and altered the composition of the intrapancreatic microbiome. In iKC mice, P. gingivalis accelerated PanIN to PDAC progression. In vitro, P. gingivalis infection induced acinar cell ADM markers SOX9 and CK19, and intracellular bacteria protected PDAC cells from reactive oxygen species-mediated cell death resulting from nutrient stress. CONCLUSION: Taken together, our findings demonstrate a causal role for P. gingivalis in pancreatic cancer development in mice.

Genome-wide DNA methylation and transcriptomic patterns of precancerous gastric cardia lesions.

BACKGROUND: Intestinal metaplasia (IM) and intraepithelial neoplasia (IEN) are considered precursors of gastric cardia cancer (GCC). Here, we investigated the histopathologic and molecular profiles of precancerous gastric cardia lesions (PGCLs) and biomarkers for risk stratification of gastric cardia IM. METHODS: We conducted a hospital-based evaluation (n = 4578) for PGCL profiles in high-incidence and non-high-incidence regions for GCC in China. We next performed 850K methylation arrays (n = 42) and RNA-seq (n = 44) in tissues with PGCLs. We then examined the protein expression of candidate biomarker using immunohistochemistry. RESULTS: Of the 4578 participants, 791 were diagnosed with PGCLs (600 IM, 62 IM with IEN, and 129 IEN). We found that individuals from high-incidence regions (26.7%) were more likely to develop PGCLs than those from non-high-incidence areas (13.5%). DNA methylation and gene expression alterations, indicated by differentially methylated probes (DMPs) and differentially expressed genes (DEGs), exhibited a progressive increase from type I IM (DMP = 210, DEG = 24), type II IM (DMP = 3402, DEG = 129), to type III IM (DMP = 3735, DEG = 328), peaking in IEN (DMP = 47 373, DEG = 2278). Three DEGs with aberrant promoter methylation were identified, shared exclusively by type III IM and IEN. Of these DEGs, we found that OLFM4 expression appears in IMs and increases remarkably in IENs (P < .001). CONCLUSIONS: We highlight that type III IM and IEN share similar epigenetic and transcriptional features in gastric cardia and propose biomarkers with potential utility in risk prediction.

Bioinformatics analyses of infiltrating immune cell participation on pancreatic ductal adenocarcinoma progression and in vivo experiment of the therapeutic effect of Shuangshen granules.

ETHNOPHARMACOLOGICAL RELEVANCE: Shuangshen granules (SSG), a nationally patented Chinese medicinal formula, including Panax quinquefolium L., Panax notoginseng (Burkill) F. H. Chen, and Cordyceps sinensis (Berk.) Sacc., has demonstrated remarkable therapeutic effects on pancreatic cancer in clinical treatment for nearly 10 years. Previous pharmacological researches have found that its main components, including ginsenosides and cordycepin have anticancer or preventive effects on pancreatic ductal adenocarcinoma (PDAC), which may be associated with immune metabolism. However, the underlying pharmacological mechanism of SSG in the truncation effect of PDAC progression is still unclear. AIM OF THE STUDY: To comprehensively understand the infiltrating immune cells during the different stages of the PDAC development chain and search for immune-related biomarkers that could potentially serve as drug targets through bioinformatic analysis. Meanwhile, the truncation effect of SSG on PDAC progression was also investigated. MATERIALS AND METHODS: The gene expression profiles at different PDAC developmental stages, including normal pancreas, pancreatic intraepithelial neoplasia (PanIN), and PDAC, were retrieved from the GEO database. The GEO2R tool was used to identify differentially expressed genes among the three groups. Functional enrichment analysis was performed with the GSEA software and Metascape platform. The CIBERSORT algorithm evaluated immune cell infiltration in the three groups, and immune-related biomarkers were identified. Correlation analysis was employed to examine the association between immune cells and the biomarkers. One of these biomarkers was selected for immunohistochemistry validation in human samples. Lastly, the effectiveness of SSG against PDAC progression and the influence on the selected biomarker were validated in vivo. The underlying pharmacological mechanisms were also explored. RESULTS: One dataset was obtained, where the functional enrichment of DEGs primarily involved immune effector processes and cytokine production of immune cells. The differential immune cells reflected during the progression from PanIN to PDAC were B memory cells, monocytes, M2 macrophages, and activated dendritic cells. The upregulation of ACTA2 was closely associated with M2 macrophage regulation. The immunohistochemistry on human samples validated significant differences in ACTA2 expression levels as the PDAC progressed. Moreover, animal experiments revealed that the national patented drug SSG ameliorated the pathological changes, decreased the expression of ACTA2 and its functional protein α-smooth muscle actin during PDAC progression. The underlying pharmacological mechanism was related to the regulation of macrophage polarization and downregulation of TGF-ß/Smad signaling pathway. CONCLUSIONS: The immunosuppressive environment changes during the PDAC progression. ACTA2 is a potential immuned-target for drug prevention of PDAC, while SSG could be a promising drug candidate.

Analytical validation and diagnostic performance of the ASCL1/ZNF582 methylation test for detection of high-grade anal intraepithelial neoplasia and anal cancer.

DNA methylation testing on biopsies can detect high-grade anal intraepithelial neoplasia (HGAIN) in need of treatment and anal cancer. This study aimed to analytically validate and determine the diagnostic performance of a newly developed multiplex quantitative methylation-specific PCR, PreCursor-M AnoGYN (RUO), combining ASCL1, ZNF582 and a reference (ACTB) in one assay. Analytical validation was performed on two qPCR devices using predefined quality criteria. Diagnostic performance was determined on a cross-sectional series of 111 anal biopsies covering all stages of anal disease. Differences in methylation levels were assessed using the Kruskal-Wallis test. Area under the curve was determined using logistic regression analysis. Detection rates were calculated at predefined specificities for the cross-sectional and an additional longitudinal series of 23 HGAIN biopsies preceding anal cancer (i.e., progressive HGAIN). For both devices analytical quality criteria were met. ASCL1 and ZNF582 methylation levels increased with increasing severity of disease (p < 6*10-8). Diagnostic performance for AIN3+ was 0.81. All cancers and virtually all progressive HGAIN were detected at 70% and 80% specificity. In conclusion, the ASCL1/ZNF582 methylation test (PreCursor-M AnoGYN (RUO)) was demonstrated to be highly robust and reproducible. Moreover, it had excellent diagnostic accuracy to detect AIN3+ and can potentially be used to guide HGAIN management.

Oncogenic KRAS, Mucin 4, and Activin A-Mediated Fibroblast Activation Cooperate for PanIN Initiation.

Over 90% of patients with pancreatic ductal adenocarcinoma (PDAC) have oncogenic KRAS mutations. Nevertheless, mutated KRAS alone is insufficient to initiate pancreatic intraepithelial neoplasia (PanIN), the precursor of PDAC. The identities of the other factors/events required to drive PanIN formation remain elusive. Here, optic-clear 3D histology is used to analyze entire pancreases of 2-week-old Pdx1-Cre; LSL-KrasG12D/+ (KC) mice to detect the earliest emergence of PanIN and observed that the occurrence is independent of physical location. Instead, it is found that the earliest PanINs overexpress Muc4 and associate with αSMA+ fibroblasts in both transgenic mice and human specimens. Mechanistically, KrasG12D/+ pancreatic cells upregulate Muc4 through genetic alterations to increase proliferation and fibroblast recruitments via Activin A secretion and consequently enhance cell transformation for PanIN formation. Inhibition of Activin A signaling using Follistatin (FST) diminishes early PanIN-associated fibroblast recruitment, effectively curtailing PanIN initiation and growth in KC mice. These findings emphasize the vital role of interactions between oncogenic KrasG12D/+ -driven genetic alterations and induced microenvironmental changes in PanIN initiation, suggesting potential avenues for early PDAC diagnostic and management approaches.

Acinar-to-Ductal Metaplasia (ADM): On the Road to Pancreatic Intraepithelial Neoplasia (PanIN) and Pancreatic Cancer.

Adult pancreatic acinar cells show high plasticity allowing them to change in their differentiation commitment. Pancreatic acinar-to-ductal metaplasia (ADM) is a cellular process in which the differentiated pancreatic acinar cells transform into duct-like cells. This process can occur as a result of cellular injury or inflammation in the pancreas. While ADM is a reversible process allowing pancreatic acinar regeneration, persistent inflammation or injury can lead to the development of pancreatic intraepithelial neoplasia (PanIN), which is a common precancerous lesion that precedes pancreatic ductal adenocarcinoma (PDAC). Several factors can contribute to the development of ADM and PanIN, including environmental factors such as obesity, chronic inflammation and genetic mutations. ADM is driven by extrinsic and intrinsic signaling. Here, we review the current knowledge on the cellular and molecular biology of ADM. Understanding the cellular and molecular mechanisms underlying ADM is critical for the development of new therapeutic strategies for pancreatitis and PDAC. Identifying the intermediate states and key molecules that regulate ADM initiation, maintenance and progression may help the development of novel preventive strategies for PDAC.

Epigenetic Alterations in DCIS Progression: What Can lncRNAs Teach Us?

Some transcripts that are not translated into proteins can be encoded by the mammalian genome. Long noncoding RNAs (lncRNAs) are noncoding RNAs that can function as decoys, scaffolds, and enhancer RNAs and can regulate other molecules, including microRNAs. Therefore, it is essential that we obtain a better understanding of the regulatory mechanisms of lncRNAs. In cancer, lncRNAs function through several mechanisms, including important biological pathways, and the abnormal expression of lncRNAs contributes to breast cancer (BC) initiation and progression. BC is the most common type of cancer among women worldwide and has a high mortality rate. Genetic and epigenetic alterations that can be regulated by lncRNAs may be related to early events of BC progression. Ductal carcinoma in situ (DCIS) is a noninvasive BC that is considered an important preinvasive BC early event because it can progress to invasive BC. Therefore, the identification of predictive biomarkers of DCIS-invasive BC progression has become increasingly important in an attempt to optimize the treatment and quality of life of patients. In this context, this review will address the current knowledge about the role of lncRNAs in DCIS and their potential contribution to the progression of DCIS to invasive BC.

Hematopoietic progenitor kinase 1 inhibits the development and progression of pancreatic intraepithelial neoplasia.

Ras plays an essential role in the development of acinar-to-ductal metaplasia (ADM) and pancreatic ductal adenocarcinoma (PDAC). However, mutant Kras is an inefficient driver for PDAC development. The mechanisms of the switching from low Ras activity to high Ras activity that are required for development and progression of pancreatic intraepithelial neoplasias (PanINs) are unclear. In this study, we found that hematopoietic progenitor kinase 1 (HPK1) was upregulated during pancreatic injury and ADM. HPK1 interacted with the SH3 domain and phosphorylated Ras GTPase-activating protein (RasGAP) and upregulated RasGAP activity. Using transgenic mouse models of HPK1 or M46, a kinase-dead mutant of HPK1, we showed that HPK1 inhibited Ras activity and its downstream signaling and regulated acinar cell plasticity. M46 promoted the development of ADM and PanINs. Expression of M46 in KrasG12D Bac mice promoted the infiltration of myeloid-derived suppressor cells and macrophages, inhibited the infiltration of T cells, and accelerated the progression of PanINs to invasive and metastatic PDAC, while HPK1 attenuated mutant Kras-driven PanIN progression. Our results showed that HPK1 plays an important role in ADM and the progression of PanINs by regulating Ras signaling. Loss of HPK1 kinase activity promotes an immunosuppressive tumor microenvironment and accelerates the progression of PanINs to PDAC.

Clinical validation of methylation biomarkers for optimal detection of high-grade vulvar intraepithelial neoplasia.

The precursor lesions of vulvar squamous cell carcinoma (VSCC) include human papillomavirus (HPV)-associated and HPV-independent squamous neoplasia with a varying cancer risk. Our study aimed to validate the accuracy of previously identified DNA methylation markers for detection of such high-grade vulvar intraepithelial neoplasia (VIN). A large clinical series of 751 vulvar lesions, originally diagnosed as high-grade VIN, were reassessed and categorized into HPV-associated or HPV-independent vulvar disease categories. Together with 113 healthy vulvar controls, all samples were tested for 12 methylation markers with quantitative multiplex methylation-specific PCR (qMSP). Performance of individual markers and selection of an optimal marker panel for detection of high-grade VIN was determined by logistic regression analysis. SST was the best-performing individual marker (AUC 0.90), detecting 80% of high-grade VIN cases, with excellent detection of HPV-independent VIN (95%), known to have the highest cancer risk. Merely 2% of controls tested methylation positive for SST. Selection of a marker panel, including ZNF582, SST and miR124-2, resulted in a comparably high accuracy for detection of high-grade VIN (AUC 0.89). In conclusion, we clinically validated the accuracy of 12 DNA methylation markers for detection of high-grade VIN. SST, as a sole marker or in a panel, provides an optimal diagnostic tool to distinguish high-grade VIN in need of treatment, particularly HPV-independent VIN, from low-grade or reactive vulvar lesions. These findings warrant further prognostic validation of methylation biomarkers for cancer risk stratification of patients with VIN.

Analysis of Donor Pancreata Defines the Transcriptomic Signature and Microenvironment of Early Neoplastic Lesions.

The adult healthy human pancreas has been poorly studied given the lack of indication to obtain tissue from the pancreas in the absence of disease and rapid postmortem degradation. We obtained pancreata from brain dead donors, thus avoiding any warm ischemia time. The 30 donors were diverse in age and race and had no known pancreas disease. Histopathologic analysis of the samples revealed pancreatic intraepithelial neoplasia (PanIN) lesions in most individuals irrespective of age. Using a combination of multiplex IHC, single-cell RNA sequencing, and spatial transcriptomics, we provide the first-ever characterization of the unique microenvironment of the adult human pancreas and of sporadic PanIN lesions. We compared healthy pancreata to pancreatic cancer and peritumoral tissue and observed distinct transcriptomic signatures in fibroblasts and, to a lesser extent, macrophages. PanIN epithelial cells from healthy pancreata were remarkably transcriptionally similar to cancer cells, suggesting that neoplastic pathways are initiated early in tumorigenesis. SIGNIFICANCE: Precursor lesions to pancreatic cancer are poorly characterized. We analyzed donor pancreata and discovered that precursor lesions are detected at a much higher rate than the incidence of pancreatic cancer, setting the stage for efforts to elucidate the microenvironmental and cell-intrinsic factors that restrain or, conversely, promote malignant progression. See related commentary by Hoffman and Dougan, p. 1288. This article is highlighted in the In This Issue feature, p. 1275.

Comprehensive DNA Methylation Analysis Indicates That Pancreatic Intraepithelial Neoplasia Lesions Are Acinar-Derived and Epigenetically Primed for Carcinogenesis.

Pancreatic ductal adenocarcinoma (PDAC) is believed to arise from the accumulation of a series of somatic mutations and is also frequently associated with pancreatic intraepithelial neoplasia (PanIN) lesions. However, there is still debate as to whether the cell type-of-origin of PanINs and PDACs in humans is acinar or ductal. As cell type identity is maintained epigenetically, DNA methylation changes during pancreatic neoplasia can provide a compelling perspective to examine this question. Here, we performed laser-capture microdissection on surgically resected specimens from 18 patients to isolate, with high purity, DNA for whole-genome bisulfite sequencing from four relevant cell types: acini, nonneoplastic ducts, PanIN lesions, and PDAC lesions. Differentially methylated regions (DMR) were identified using two complementary analytical approaches: bsseq, which identifies any DMRs but is particularly useful for large block-like DMRs, and informME, which profiles the potential energy landscape across the genome and is particularly useful for identifying differential methylation entropy. Both global methylation profiles and block DMRs clearly implicated an acinar origin for PanINs. At the gene level, PanIN lesions exhibited an intermediate acinar-ductal phenotype resembling acinar-to-ductal metaplasia. In 97.6% of PanIN-specific DMRs, PanIN lesions had an intermediate methylation level between normal and PDAC, which suggests from an information theory perspective that PanIN lesions are epigenetically primed to progress to PDAC. Thus, epigenomic analysis complements histopathology to define molecular progression toward PDAC. The shared epigenetic lineage between PanIN and PDAC lesions could provide an opportunity for prevention by targeting aberrantly methylated progression-related genes. SIGNIFICANCE: Analysis of DNA methylation landscapes provides insights into the cell-of-origin of PanIN lesions, clarifies the role of PanIN lesions as metaplastic precursors to human PDAC, and suggests potential targets for chemoprevention.