The Journal of Pathology

BackgroundIntraductal papillary mucinous neoplasms (IPMNs) are preinvasive pancreatic lesions with a spectrum of histologic phenotypes and variable risk in progressing to invasive cancer. Aberrant repetitive element expression has been shown to be functionally linked to cell state changes in pancreatic cancer. ObjectiveThis study utilized spatial transcriptomics with customized repeat element probes to better understand the relationship of histologic subtypes, repeat element dysregulation, and molecular profiles of different cell populations in IPMN. DesignA total of 52 lesions from 18 patients with resected IPMNs of different histologies and degrees of dysplasia were analyzed with whole transcriptome spatial analysis (GeoMx). Of these, 50 lesions from 17 patients were also processed for single-cell spatial molecular imaging (CosMx). Repeat element probes for LINE1, HSATII, HERVK, and HERVH were used for GeoMx and CosMx. ResultsPancreaticobiliary-type IPMN was enriched for basal-like epithelium and infiltration of Treg cells. Intestinal-type IPMN was enriched for classical epithelium and macrophage infiltrates. Gastric-type IPMN was found to have equal basal-like and classical epithelium with a diverse immune infiltrate. Repeat RNAs were expressed at high levels across IPMN phenotypes and enriched in high-grade dysplasia. Single-cell transcriptional trajectory analysis revealed a phylogeny starting from gastric toward intestinal and pancreaticobiliary branches associated with higher-grade dysplasia and repeat RNA expression. ConclusionSpatial transcriptomics of IPMN identified a molecular continuum between histological subtypes supporting a common gastric-type origin that transitions to intestinal and pancreatobiliary phenotypes. This cell state plasticity is linked with repeat element expression that can be a potential biomarker for IPMN progression. What is already known on this topicO_LIMolecular characterization of IPMN subtypes using regional spatial transcriptomics has described the differences between histologies C_LIO_LIRepeat element expression is associated with cell state changes in pancreatic ductal neoplasm C_LI What this study addsO_LISingle cell spatial molecular imaging of IPMN subtypes reveals a cellular and molecular continuum starting from gastric histology with distinct branches to Intestinal and pancreatobiliary subtypes C_LIO_LIRepeat element expression is associated with higher grade IPMN histology C_LIO_LIRepeat element expression is elevated in the histologic and molecular continuum from gastric to intestinal and pancreatobiliary-type IPMN C_LI How this study might affect research, practice, or policyO_LIThese results support a common gastric histology origin of IPMN subtypes with different molecular trajectories towards higher grade disease C_LIO_LIOur findings suggest that repeat RNA expression can be used in conjunction with other transcriptional markers of cell state as a biomarker for IPMN progression C_LI

PurposeIntraductal papillary mucinous neoplasms (IPMN) occur in 5-10% of the population, but only a small minority progress to pancreatic ductal adenocarcinoma (PDAC). The lack of accurate predictors of high-risk disease leads both to unnecessary operations for indolent neoplasms as well as missed diagnoses of PDAC. Digital spatial RNA profiling (DSP-RNA) provides an opportunity to define and associate transcriptomic states with cancer risk. Experimental DesignWhole-transcriptome DSP-RNA profiling was performed on 10 IPMN specimens encompassing the spectrum of dysplastic changes from normal duct to cancer. Ductal epithelial regions within each tissue were annotated as normal duct (NL), low-grade dysplasia (LGD), high-grade dysplasia (HGD), or invasive carcinoma (INV). Gene expression count data was generated by Illumina sequencing and analyzed with R/Bioconductor. ResultsDimension reduction analysis exposed three clusters reflecting IPMN transcriptomic states denoted "normal-like" (cNL), "low-risk" (cLR) and "high-risk" (cHR). In addition to specific marker genes, the three states exhibited significant enrichment for the exocrine, classical, and basal-like programs in PDAC. Specifically, exocrine function diminished in cHR, classical activation distinguished neoplasia from cNL, and basal-like genes were specifically upregulated in cHR. Intriguingly, markers of cHR were detected in NL and LGD regions from specimens with PDAC but not low-grade IPMN. ConclusionsDSP-RNA of IPMN revealed low-risk (indolent) and high-risk (malignant) expression programs that correlated with the activity of exocrine and basal-like PDAC signatures, respectively, and distinguished pathologically low-grade from malignant specimens. These findings contextualize IPMN pathogenesis and have the potential to transform existing risk stratification models. Statement of translational relevanceCurrent consensus guidelines for management of intraductal papillary mucinous neoplasms (IPMN) of the pancreas utilize clinical and radiographic criteria for risk stratification. Unfortunately, the estimated positive predictive value of these criteria for IPMN-associated pancreatic ductal adenocarcinoma (PDAC) is under 50%, indicating that over half of pancreatectomies are performed for benign disease. Moreover, nearly 15% of patients who were deemed "low risk" by the same criteria harbored PDAC. Surgical resection of IPMN has maximal benefit when performed prior to the development of PDAC, as evidence of carcinoma has been associated with a high rate of recurrence and poor overall survival. Thus, the development of molecular diagnostics that improve the accuracy of IPMN risk classification would have immediate relevance for patient care, both in terms of better selecting patients for potentially curative operations, as well as sparing patients with low-risk lesions from invasive procedures.

By a classical approach, the invasive margins of colorectal carcinomas can be typed as expansive or infiltrative, the latter portending a poor prognosis. Colorectal carcinomas with infiltrative-type invasive margins have long been known for their peculiar stroma and the relative absence of a lymphohistiocytic host response, but they have not been studied with contemporary gene expression techniques. Therefore, we submitted 18 microsatellite-stable colorectal carcinomas that could be typed as having invasive margins of the infiltrative (N = 7) or expansive type (N = 11) with confidence to nanoString nCounter(R) analysis (Tumor Signaling 360 Panel). Tissue microarray punch samples were obtained from the tumors and prior to RNA extraction histological sections were prepared. The invasive margin types were mirrored surprisingly well in the two main clusters delineated by an unsupervised cluster analysis of the gene expression data, but tumor budding, the second type of colorectal carcinoma invasion phenotype, was not. Nanostring Annotation Scores were significant for signaling pathways (TGFb, PDGF, MET, FGFR), extracellular matrix remodeling, and anti-tumor immunity processes. However, any hopes that the tumour biology behind the two phenotypes of invasion could be pinpointed to differential expressions of a small set of genes were not fulfilled. Taken together, the data indeed give a molecular underpinning to the two invasion phenotypes, pointing out that matrix features and anti-tumor immunity are key. Nevertheless, we failed to gain a more detailed insight into the mechanics at work, and this may well be due to general limitations of the technology employed.

Intraductal papillary mucinous neoplasms (IPMNs) are cystic precursor lesions to pancreatic ductal adenocarcinoma (PDAC). IPMNs undergo multistep progression from low grade (LG) to high grade (HG) dysplasia, culminating in invasive neoplasia. While patterns of IPMN progression have been analyzed using multi-region sequencing for somatic mutations, there is no integrated assessment of molecular events, including copy number alterations (CNAs) and transcriptomics changes, that accompany IPMN progression. We performed laser capture microdissection on surgically resected IPMNs of varying grades of histological dysplasia obtained from 24 patients (total of 74 independent histological lesions), followed by whole exome and whole transcriptome sequencing. Overall, HG IPMNs displayed a significantly greater aneuploidy score than LG lesions, with chromosome 1q amplification, in particular, being associated with HG progression and with cases that harbored cooccurring PDAC. Furthermore, the combined assessment of single nucleotide variants (SNVs) and CNAs identified both linear and branched evolutionary trajectories, underscoring the heterogeneity in the progression of LG lesions to HG and PDAC. At the transcriptome level, upregulation of MYC-regulated targets and downregulation of transcripts associated with the MHC class I antigen presentation machinery was a common feature of progression to HG. Taken together, this work emphasizes the role of 1q copy number amplification as a putative biomarker of high-risk IPMNs, underscores the importance of immune evasion even in non-invasive precursor lesions, and supports a previously underappreciated role of CNA-driven branching evolution as an avenue for IPMN progression. Our study provides important molecular context for risk stratification and cancer interception opportunities in IPMNs. SignificanceIntegrated molecular analysis of genomic and transcriptomic alterations in the multistep progression of intraductal papillary mucinous neoplasms (IPMNs), which are bona fide precursors of pancreatic cancer, identifies features associated with progression of low-risk lesions to high-risk lesions and cancer, which might enable patient stratification and cancer interception strategies.

Intraductal papillary mucinous neoplasm (IPMN) represents one type of pancreatic ductal adenocarcinoma (PDA) precursor lesion, however its cell-of-origin remains unclear. Here we describe a new mouse model in which pancreas-specific Cre activation of a nuclear glycogen synthase kinase-3{beta} transgene is combined with oncogenic KRas (referred to as KNGC). KNGC mice show accumulation of neoplastic ductal cells at 4-weeks that progressively develop into IPMN with low-grade dysplasia in advanced age. RNA-sequencing identified expression of several terminal duct cell lineage genes including Agr2 and Aqp5. Interestingly, Aqp5, a water channel, was found to be required for the development of IPMN lesions in KNGC mice. Staining of human IPMN samples indicates that these preneoplastic lesions also arise from expansion of the terminal duct population. Altogether, these data highlight the utility of the KNGC model for understanding the biology of IPMN and potential utility in defining predictive biomarkers of IPMN - PDA development. Statement of significanceUnderstanding the cell-of-origin of IPMN is crucial to developing early detection methods that specifically target aggressive precursors of PDA. This work, using a novel mouse model, identifies Aqp5-modulated development of Agr2+ terminal ducts that could potentially serve as a clinical biomarker for IPMN.

BackgroundIntraductal papillary mucinous neoplasms (IPMNs) are clinically detectable precursors of pancreatic adenocarcinoma, yet the mechanisms initiating their development remain poorly defined. Although KRAS mutations are highly frequent in human IPMNs, KRAS activation in pancreatic ductal cells alone fails to recapitulate IPMN development in murine models, indicating that additional tumor-suppressive mechanisms must be overcome. ObjectiveThe objective was to determine whether loss of the transcription factor HNF1B predisposes to initiation of IPMN. DesignWe assessed HNF1B nuclear expression and promoter methylation in resected human IPMN specimens. To model IPMN initiation, we generated mice with ductal-specific inactivation of Hnf1b, alone or combined with KRASG12D. Ductal organoids and RNA-sequencing were used to investigate molecular mechanisms. Transcriptomic analyses were also performed on human IPMN surgical specimens. MRI from germline HNF1B mutation/deletion carriers was re-evaluated for IPMN prevalence. ResultsHuman IPMNs showed loss of HNF1B by immunochemistry, with enrichment to promoter methylation that increased with dysplasia grade. The KHC model recapitulated the key features of IPMN development including ductal dilation, high proliferation, papillary architecture and mucin production. Loss of Hnf1b together with Kras activation induced loss of primary cilia, cellular reprogramming and engaged oncogenic YAP and Wnt/{beta}-catenin signaling, similar to human IPMNs. Moreover, germline HNF1B carriers exhibited a markedly increased prevalence of branch-duct IPMN. ConclusionHNF1B functions as a tumor-suppressive gatekeeper of pancreatic ductal cells. These findings highlight HNF1B inactivation as a potential biomarker and therapeutic entry point for early interception of IPMN-driven pancreatic cancer. They also have implications for the surveillance of HNF1B-syndrome.

IntroductionBreast cancer, a major contributor to cancer-related deaths in females, presents significant challenges in classification and diagnosis, particularly triple negative breast cancers (TNBC). This study explores the utility of Trichorhinophalangeal syndrome type 1 (TRPS1) as a novel biomarker, comparing its efficacy with GATA3 in TNBC with a specific focus on spindle cell neoplasms. MethodsOur pathology database was searched to identify breast carcinoma cases between January 2021 and July 2021. All cases of poorly differentiated breast carcinoma (primary and metastatic) and primary spindle cell neoplasms of breast confirmed on core biopsies, in women greater than 18 years were selected. Immunohistochemical staining for ER, PR, HER2, GATA3 and TRPS1 were interpreted by two pathologists. TRPS1 and GATA3 expression levels among different groups were analyzed. ResultsOur study cohort comprised of 60 cases (24 primary breast carcinomas, 22 metastatic breast carcinomas and 14 spindle cell lesions of the breast). Our study revealed TRPS1s superior sensitivity in TNBC, with 84% positivity compared to 53% for GATA3. TRPS1 showed promise in diagnosing spindle cell neoplasms, identifying 6/14 (43%) of cases, while GATA3 yielded no positive results. The six spindle cell cases that were TRPS1 positive were one synovial sarcoma, two malignant phyllodes, one fibromatosis, one myofibroblastoma and one angiosarcoma. ConclusionTRPS1 holds substantial promise as a sensitive and novel diagnostic marker for TNBC in comparison to GATA3. The differential diagnostic utility of TRPS1 warrants careful consideration due to its positivity in other spindle cell neoplasms of the breast, which may present as differential diagnoses for metaplastic carcinoma. HighlightsO_LITRPS1 demonstrated superior sensitivity (84%) compared to GATA3 (53%) in identifying triple-negative breast carcinomas (TNBC), supporting its role as a promising diagnostic marker. C_LIO_LITRPS1, unlike GATA3, showed positivity in 43% of spindle cell breast neoplasms, including malignant phyllodes, fibromatosis, myofibroblastoma, angiosarcoma, and synovial sarcoma, highlighting its broader expression spectrum. C_LIO_LIPathologists should exercise caution in interpreting TRPS1 positivity within spindle cell lesions, as its expression may mimic metaplastic carcinoma, underscoring the need for careful correlation with morphology and other markers. C_LI

PurposeReliable prediction of invasive progression remains an unmet need in ductal carcinoma in situ (DCIS). Conventional histologic grading provides limited prognostic value. Building on mechanistic evidence that cell crowding in confined ducts engages a TRPV4-dependent mechanotransduction program, we evaluated plasma-membrane TRPV4 (PM-TRPV4) relocalization as a mechanosensitive, patient-level prognostic biomarker for progression to invasive ductal carcinoma (IDC). Experimental DesignIn a retrospective single-institution case-control study, we analyzed 44 patients with pure DCIS (24 progressed to IDC, 20 remained IDC-free [≥]5 years). Three board-certified pathologists independently scored PM-TRPV4 on whole-slide immunohistochemistry using a prespecified five-level membrane-localization rubric (linearly weighted Fleiss {kappa}=0.823, 95% CI 0.777-0.863). The primary endpoint was IDC-free survival assessed by Kaplan-Meier and Cox models with prespecified adjustment for histologic grade and estrogen receptor (ER) status. ResultsPM-TRPV4 positivity was associated with shorter IDC-free survival (log-rank p=0.040). In multivariable Cox models, PM-TRPV4 remained independently prognostic (adjusted HR=3.77; 95% CI 1.01-14.12; p=0.049) after accounting for grade and ER, whereas histologic grade itself was not prognostic (p=0.57). Restricting to lower-grade DCIS strengthened the association (OR=10.50, two-sided p=0.016) vs overall (OR=5.92, two-sided p=0.057). In lower-grade DCIS, 77.8% (14/18) of PM-TRPV4-positive cases progressed vs 25.0% (2/8) of PM-TRPV4-negative. ConclusionsPM-TRPV4 provides independent, mechanism-informed prognostic information beyond histologic grade and ER. Given the small, single-institution case-control design, findings are hypothesis-generating and support multi-institutional validation with standardized staining/scoring, external calibration, and [≥]10 years of surveillance, particularly to refine risk in lower-grade DCIS, where clinical decisions are most uncertain. Translational Relevance StatementCurrent DCIS management relies on histologic grade, which has variable reproducibility and offers little guidance for about 60% of patients with lower-grade disease. We show that plasma-membrane relocalization of TRPV4 (PM-TRPV4), a functional readout of pro-invasive mechanotransduction in crowded ducts, adds independent prognostic information beyond grade and ER. In our cohort (N=44; 24 events), PM-TRPV4 positivity was associated with shorter IDC-free survival and remained significant in multivariable models (adjusted HR 3.77; p=0.049), whereas grade was not prognostic. Exploratory analyses suggested larger effects in lower-grade DCIS (OR [~]10.5), identifying a subset at high risk (77.8% vs 25.0% progression). PM-TRPV4 achieved near-perfect inter-rater reliability (weighted kappa = 0.823) using standard IHC on routine FFPE sections, supporting practical deployent. If externally validated with standardized protocols and at least 10 years of surveillance, PM-TRPV4 testing could enable personalized risk stratification and help select lower-grade DCIS patients for active surveillance versus those requiring escalation.

AimsThis cross-sectional study aims to identify potential transcriptomic changes conveyed by presence of amyloid deposits in islets from pancreatic tissue obtained from metabolically profiled living donors. MethodsAfter establishing Thioflavin S as the most sensitive approach to detect islet amyloid plaques, we utilized RNA sequencing data obtained from laser capture microdissected islets to define transcriptomic effects of this pathological entity. The RNA sequencing data was used to identify differentially expressed genes by linear modeling. Further analyses included functional enrichment analysis of KEGG and Hallmark gene sets as well as a weighted gene correlation network analysis. ResultsEleven differentially expressed genes were identified in islets affected by amyloidosis. Enrichment analyses pointed to signatures related to protein aggregation diseases, energy metabolism and inflammatory response. A gene co-expression module was identified that correlated to islet amyloidosis. ConclusionAlthough the influence of underlying Type 2 diabetes could not be entirely excluded, this study presents a valuable insight into the biology of islet amyloidosis, particularly providing hints into the potential relationship between islet amyloid deposition and structural and functional proteins involved in insulin secretion. Research in contextO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIIslet amyloidosis is the only histological marker of Type 2 diabetes in the pancreas C_LIO_LIIndividuals not suffering from Type 2 diabetes can also be affected by islet amyloidosis C_LIO_LIThe clinicopathological significance of this phenomenon is still unclear C_LI What is the key question?O_LIDoes the islet transcriptome of individuals with islet amyloidosis provide explanations for the onset of this phenomenon and its pathophysiological value? C_LI What are the new findings?O_LIIslet transcriptomes of affected subjects exhibit only limited transcriptomic differences compared to unaffected ones. C_LIO_LIStructural and functional proteins involved in insulin secretion machinery may be involved in the pathophysiological sequence of amyloid formation C_LI

Non-destructive 3D pathology methods have emerged in recent years with the potential to enhance standard 2D histopathology by greatly increasing the amount of tissue sampled by imaging and by providing volumetric morphological context. Another key advantage is that tissues remain intact, allowing re-embedding after imaging for potential long-term storage and future histological or molecular analyses. However, the impact of 3D pathology protocols on biomolecules -- including DNA, RNA, and proteins -- and their compatibility with downstream assays, has not been systematically evaluated. Here, we applied a previously optimized 3D pathology protocol -- involving deparaffinization, fluorescent H&E-analog staining, optical clearing, and open-top light-sheet microscopy -- to formalin-fixed paraffin-embedded (FFPE) specimens of breast, prostate, and head and neck cancer. Following the protocol, tissues were re-embedded in paraffin and compared with paired FFPE controls that did not undergo 3D pathology processing. DNA and RNA were extracted and subjected to quality assessments. Amplifiability was tested by PCR and reverse transcription quantitative PCR (RT-qPCR) of housekeeping genes. Although the results showed a slight decrease in the average yield and increased fragmentation of both DNA and RNA, amplifiability was largely preserved. Sanger sequencing of the PCR products confirmed accurate sequence determinations, while total RNA sequencing indicated that the global transcriptomic profile was largely unchanged. IHC staining of common biomarkers produced comparable signals, suggesting those proteins are well preserved after the 3D pathology workflow. These results demonstrate the feasibility of combining 3D pathology with downstream molecular applications.

Atypical fibroxanthoma (AFX) and pleomorphic dermal sarcoma (PDS) are cutaneous neoplasms that fall along a spectrum. PDS is more aggressive than AFX with higher rates of local and distant metastases. Diagnostic biomarkers for AFX and PDS are lacking and therefore these tumors are diagnosed only after excluding other dermal spindle cell neoplasms, including cutaneous leiomyosarcoma (cLMS), spindle cell melanoma (SCM), and sarcomatoid squamous cell carcinoma (sSCC). To identify clinically valuable biomarkers, we contrast the tumors within the diagnostic differential using single-cell RNA sequencing and bulk proteomic data. Gene Ontology (GO) analysis of transcripts and proteins enriched in AFX/PDS identified multiple shared pathways associated with cell adherence and the extracellular matrix. We identify that LRP1, LTBP2, and NAV1 are all enriched in AFX/PDS over other tumors in the differential at both the level of mRNA and protein. IHC reveals that LRP1 is 90% sensitive and 73% specific for AFX/PDS in a cohort of AFX, PDS, cLMS, SCM, and sSCC. This outperforms published data for CD10, which is currently used clinically (sensitivity 83.5% and specificity 50%). When used in conjunction with LTBP2, specificity for AFX/PDS within the differential rises from 73% to 93%. These findings suggest that LRP1, particularly if evaluated in conjunction with existing stains, can improve diagnostic accuracy for AFX and PDS.

ContextWhile fibroadenomas (FAs) are the most common benign neoplasm of the breast, little data exists about the clinical characteristics and prognostic value of FAs found to be directly associated with atypical and malignant lesions. ObjectiveCases of FA of the breast were reviewed to establish the exact clinical significance of these lesions involved by atypia and malignancy. DesignAll FA cases diagnosed on core needle biopsy (CNB) between 2013 and 2022 were screened to identify atypical and malignant lesions {(atypical lobular hyperplasia (ALH), lobular carcinoma in situ (LCIS), atypical ductal hyperplasia (ADH), flat epithelial atypia (FEA), ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), and invasive lobular carcinoma (ILC)} arising in FA. The relationship between CNB and excisional findings for each case was reviewed. ResultsA total of 1500 cases of FA diagnosed on CNB were identified. Among these, 23 cases showed atypical and/or malignant lesions within FA. The median age at diagnosis was 53 years. Of those 23 cases, five were LCIS, one was LCIS+ILC, one was LCIS+ALH, one DCIS+IDC, one was LCIS+DCIS, three were ADH, seven were ALH, and four were DCIS. For LCIS, the excision showed LCIS (2/5), LCIS + ALH (1/5), LCIS + DCIS (1/5), LCIS + DCIS + ALH (1/5). For DCIS, excisions showed Invasive carcinoma with mixed ductal and lobular features (1/4), LCIS + DCIS (1/4), LCIS + DCIS + IDC [classic and pleomorphic type] (1/4), no residual carcinoma (1/4). For ALH, excision showed ALH (2/7), no residual ALH (2/7), ALH+IDP (Intraductal Papilloma) (2/7), LCIS (1/7). For ADH, DCIS (1/3), and benign findings (2/3). For LCIS+ILC and DCIS+IDC, the excisional findings were the same. For LCIS+ ALH, the excisional findings showed benign findings with the radial scar (1/1). ConclusionThere is a low percentage of FA harboring atypia or carcinoma. Due to the high upgrade rate following excision, complete excision of these lesions may guide recommended method of clinical management

Collagen organisation within the tumour microenvironment plays a critical role in tumour progression and has emerged as an important structural biomarker in cancer. Second Harmonic Generation (SHG) microscopy enables label-free visualisation and quantitative assessment of fibrillar collagen architecture; however, its high cost, specialised instrumentation, and limited field-of-view restrict routine clinical application. In this study, we evaluated whether collagen features quantified from digitally scanned Masson-Goldners Trichrome-stained histopathological sections can approximate measurements obtained from SHG microscopy. Formalin-fixed paraffin-embedded breast tumour tissues, including benign and invasive ductal carcinoma (IDC) samples with varying collagen content, were analysed using SHG microscopy and whole-slide brightfield imaging. Matched regions of interest were analysed using two independent digital image analysis approaches: a conventional ImageJ-based workflow (TWOMBLI) and a machine learning-based computational pipeline. Collagen structural parameters including collagen deposition area, fibre number, and alignment metrics were quantified and compared across imaging modalities using correlation analysis. SHG signals were consistently detected from trichrome-stained sections, confirming compatibility of SHG imaging. Quantitative comparison demonstrated significant concordance between SHG-derived collagen metrics and those obtained from digital image analysis pipelines, particularly for collagen area and fibre alignment. These findings demonstrate that computational analysis of routine histopathological images can capture key spatial features of collagen organisation comparable to SHG microscopy. Digital pathology-based collagen quantification therefore, represents a scalable and clinically accessible approach for assessing extracellular matrix architecture in tumour tissues.

Pigmented paraganglioid carcinoid tumors (PPCT) of the lung are a rare, underrecognized, and poorly characterized morphologic variant of pulmonary neuroendocrine tumors (NETs). While these tumors are usually diagnosed as typical carcinoid (TC) tumors, PPCT may represent a diagnostic challenge due to the histologic resemblance with extra-adrenal paraganglioma (PG). In this study, we aimed to comprehensively characterize the histomorphologic, immunophenotypic, and transcriptomic profiles of PPCT in comparison to TC and PG using spatially resolved transcriptomic analysis. Using a tissue microarray (TMA) composed of 38 tumors, including 20 TC, 16 PG, and 2 PPCT, we performed immunohistochemical (IHC) and digital spatial transcriptomic (GeoMx(R) DSP) profiling. The TMA included two punches and two regions of interest (ROIs) per case. Cellular transcriptomes were selected based on epithelial (PanCK+), sustentacular (S100+), and immune (CD45+) compartments. By IHC, PPCT retained neuroendocrine markers (synaptophysin, INSM1, chromogranin A) but showed decreased or absent pancytokeratin cocktail expression and increased number of sustentacular cells highlighted by strong expression of S100 and SOX-10, similar to PG. Expression of AE1/AE3 and CK8/18 confirmed their epithelial origin and helped distinguish them from PG. The transcriptome of PPCT clustered with that of TC but displayed distinct expression patterns in a small subset of genes. Although the sustentacular and immune compartments showed limited divergence, the epithelial compartment showed differentially expressed genes in PPCT including FABP5, MLPH, GPNMB, and SOX1, which indicate upregulation of melanocytic and neural crest markers. Gene set enrichment analysis (GSEA) revealed significant upregulation of pathways related to inflammation (e.g., TLR4-TRAF6-TAK1), PTEN trafficking, and inositol phosphate metabolism. PPCT show increased melanocytic pathway expression, which may explain the morphologic resemblance to PG.

BACKGROUND & AIMSIntraductal Papillary Mucinous Neoplasms (IPMNs) are cystic lesions and bona fide precursors for pancreatic ductal adenocarcinoma (PDAC). Recent studies have shown that pancreatic precancer is characterized by a transcriptomic program similar to gastric metaplasia. The aims of this study were to assay IPMN for pyloric markers, to identify molecular drivers, and to determine a functional role for this program in the pancreas. METHODSPyloric marker expression was evaluated by RNA-seq and multiplex immunostaining in patient samples. Cell lines and organoids expressing KrasG12D +/- GNASR201C underwent RNA sequencing. A PyScenic-based regulon analysis was performed to identify molecular drivers, and candidates were evaluated by RNA-seq, immunostaining, and small interfering RNA knockdown. Glycosylation profiling was performed to identify GNASR201C-driven changes. Glycan abundance was evaluated in patient samples. RESULTSPyloric markers were identified in human IPMN. GNASR201Cdrove expression of this program as well as an indolent phenotype characterized by distinct glycosyltransferase changes. Glycan profiling identified an increase in LacdiNAcs and loss of pro-tumorigenic Lewis antigens. Knockdown of transcription factors Spdef or Creb3l1 or chitinase treatment reduced LacdiNAc deposition and reversed the indolent phenotype. LacdiNAc and 3-sulfoLeA/C abundance discriminated low from high grade patient IPMN. CONCLUSIONGNASR201C drives an indolent phenotype in IPMN by amplifying a differentiated, pyloric phenotype through SPDEF/CREB3L1 which is characterized by distinct glycans. Acting as a glycan rheostat, mutant GNAS elevates LacdiNAcs at the expense of pro-tumorigenic acidic Lewis epitopes, inhibiting cancer cell invasion and disease progression. LacdiNAc and 3-Sulfo-LeA/C are mutually exclusive and may serve as markers of disease progression.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers due to late diagnosis and poor therapeutic efficiency. Throughout PDAC progression, a dense extracellular matrix (ECM) is deposited around neoplastic cells and accompanies tumor development and aggressiveness. This significant stroma, both in terms of quantity and through its impact on tumor cells, is now considered as a target for innovative therapies to improve patient survival. Among ECM proteins, Tenascins (TNs) are a family of four glycoproteins (TNC, TNW, TNR and TNX) sharing a common modular structure, but exhibiting different expression patterns and functions depending on the physiological and physio-pathological contexts. In PDAC, TNC is up-regulated and is considered as a pro-tumorigenic actor, whereas TNXs role remains to be elucidated. Herein, we demonstrated that unlike TNC, TNX is drastically decreased in PDAC, and that this loss is correlated with reduced patient survival. Dysregulation of the TNX/TNC balance is attributable to Transforming Growth Factor-beta (TGF-{beta}) upregulation during pancreatic carcinogenesis. Interestingly, we found that TNX is first highly deposited in low grade lesions before being clearly decreased in later stages suggesting an elaborate stromal remodelling during PDAC development. Finally, low TNX and high TNC deposition around precursor lesions are correlated with increased preneoplastic cell proliferation. Altogether, our results (i) demonstrate the importance of Tenascin ratios during pancreatic carcinogenesis, (ii) suggest an anti-proliferative role for TNX unlike its pro-tumoral counterpart, TNC and (iii) underscore TNX and TNC as valuable targets for the development of new drugs for pancreatic cancer and/or solid tumor treatment. Statement of significanceThe tumor microenvironment plays a prominent but contradictory role in pancreatic cancer. Our study highlights the role of a TGF-{beta}-controlled TNX/TNC balance and designates TNX as an inhibitor of tumor cell proliferation.

BackgroundPatients demonstrating strong immune responses to primary colorectal cancer (CRC) have a survival benefit following surgery, while those with predominantly stromal microenvironments do poorly. Biomarkers to identify patients with colorectal cancer liver metastases (CRLM) who have good prognosis following surgery for oligometastatic disease remain elusive. The aim of this study was to determine the practical application of a simple histological assessment of immune cell infiltration and stromal content in predicting outcome following synchronous resection of primary CRC and CRLM, and to interrogate the underlying functional biology that drives disease progression. MethodsPatients undergoing synchronous resection of primary CRC and CRLM underwent detailed histological assessment, panel genomic and bulk transcriptomic assessment, immunohistochemistry (IHC) and GeoMx Spatial Transcriptomics (ST) analysis. Integration with genomic features, pathway enrichment analysis and immune deconvolution were performed. ResultsHigh-immune metastases were associated with improved cancer specific survival (HR, 0.36, P=0.01). Bulk transcriptomic analysis was confounded by stromal content but ST demonstrated that the invasive edge of the metastases of long-term survivors was characterized by adaptive immune cell populations enriched for Type II Interferon signalling (NES=-2.05 P.Adj<0.005) and MHC-Class II Antigen Presentation (NES=-2.09 P.Adj<0.005). In contrast, patients with poor prognosis demonstrated increased abundance of regulatory T-cells and neutrophils with enrichment of Notch (NES=2.2 P.Adj=0.022) and TGF-{beta} (NES=2.2 P.Adj=0.02) signalling pathways at the metastatic tumor centre. ConclusionsHistological assessment stratifies outcome in patients undergoing synchronous resection of CRLM. ST analysis reveals significant intra-tumoral and inter-lesional heterogeneity with underlying transcriptomic programmes identified in driving each phenotype. TRANSLATIONAL RELEVANCEThe current study demonstrates that accurate histological assessment of immune cell infiltration and stromal content can define survival in patients following resection of oligometastatic liver disease when presenting synchronously with primary colorectal cancer. A spatial transcriptomic approach has demonstrated heterogeneity between patients, between matched lesions in the same patient and within individual lesions. Patients with high immune infiltrates at the invasive margin demonstrated lymphocytic infiltration and associated upregulated adaptive immune pathways in long term survivors. In specimens with low immune infiltrate at the tumor edge a significant reduction in survival was observed, this was determined by upregulated immunosuppressive pathways and a predominance of innate immune cells surrounding metastases. Spatial transcriptomics can be used to examine drivers of metastatic progression in CRC and identifies patients with reactive and suppressed immune microenvironments. Application across a larger cohort will build the cartography of CRLM, while in future, studies may assess application of this technology to pre and post treatment biopsy samples with the aim of predicting individual therapeutic responses. The current study has highlighted discrepancies between bulk and ST derived data whilst demonstrating accuracy of deconvoluted transcriptome to determine immune profiling. Now that ST strategies are becoming more achievable at scale, this has implications for the interpretation of the bulk transcriptomic signatures both of primary and metastatic CRC.

PurposeUterine leiomyomas are common benign tumors that arise from smooth muscle and can significantly impact quality of life. Over the past two decades, uterine artery embolization has risen as a minimally invasive alternative treatment to hysterectomy or myomectomy for the management of leiomyomas. While prior work has established the safety of this procedure, there exist few reports quantifying sequelae, notably rates of subsequent treatment and primary ovarian insufficiency. The purpose of this study is to demonstrate the efficacy of uterine artery embolization as well as to investigate the frequency of gynecologic reintervention and primary ovarian insufficiency following treatment. MethodsThe study cohort consisted of patients (n=199) who presented with symptoms concerning for uterine pathology and with leiomyoma(s) confirmed by MRI. This cohort underwent embolization between January 2013 and December 2018 at a single academic institution. Data was collected from retrospective chart review and included demographics, symptomology, imaging, procedural details, and follow-up care. This data was subsequently analyzed to quantify the frequencies of various outcomes at 4-10 years following embolization. ResultsOf 199 symptomatic patients with confirmed leiomyomas, all underwent technically successful uterine artery embolization. At the time of follow-up, information was available for 188 (94.5%) patients, of which 145 (77.1%) reported significant symptomatic improvement while 34 required additional intervention--either medical (9%) or surgical (9%). The most common secondary medical management involved hormone therapy, while the most common subsequent gynecologic procedure was a hysterectomy. Additionally, there were seven (3.7%) cases of amenorrhea following embolization. ConclusionGiven its minimally invasive nature, rapid recovery time, and uterine-sparing capability, uterine artery embolization should be considered a frontline therapy for symptomatic leiomyomas. This study supports an overall low complication rate, limited hospitalization time, near-complete resolution of symptoms, and low risk of ovarian dysfunction for a majority of patients. Following embolization, only 9% of patients required additional medical management, and only 9% required a second procedural intervention.

BackgroundBRCA1-associated protein 1 (BAP1) is a deubiquitinase, frequently altered in cancers including hepatocellular carcinoma and cholangiocarcinoma. While Bap1 has been shown to play key roles in metabolism, maintenance of tissue homeostasis, and immune cell development, little is known about its normal functions in the liver in vivo. This study aims to identify Bap1 specific effects on the livers immune microenvironment and biological functions. MethodsUsing AAV8-mediated CRISPR/CAS9 genome editing we generated a mouse hepatocyte-specific model of Bap1 knockout to define the changes that occur in liver biology in an in vivo system and characterize how loss of Bap1 alters the livers response to injury. Single-cell resolution spatial transcriptomics were performed in conjunction with immunohistochemistry to analyze cell-type composition and immune cell recruitment changes. Bulk RNA-sequencing was performed for further assessment of the impact of Bap1 loss on transcription. ResultsHepatocyte-specific depletion of Bap1 induced transcriptional changes shared with acute injury. We observed a strong dysregulation of inflammatory pathways associated with BAP1 loss. Moreover, the transcriptional response of Bap1 depletion in hepatocytes to damage was markedly different than in control liver, with Bap1-deleted livers showing a decreased hepatocyte identity based on gene expression. Spatial transcriptomics and quantitative texture analysis of immunohistochemistry revealed an altered immune environment prior to damage and an impaired recruitment of immune cells in Bap1 depleted livers after damage. ConclusionsUsing a hepatocyte-specific Bap1 deletion we identified Bap1 as a critical modulator in the livers immune cell response. We show that Bap1 loss leads to an inflammatory environment prior to damage and disrupts the recruitment immune cells. Our quantitative spatial analysis highlights the power of such approaches to characterize the spatial distribution of different cell types in a tissue.

We investigated the association between pancreatic ductal adenocarcinoma (PDAC) subtypes and neural invasion (NI) status in order to assess what factors influence the inter-tumor heterogeneity of NI. We built single-sample classifiers for prominent subtype schemes using multiple approaches. We predicted the subtype labels of samples with known NI status using the classifiers based on pairwise gene expression comparisons, which were consistently the best-performing models. These classifiers were made available in a Shiny app (https://salihaey.shinyapps.io/pcsubtypes/). Stromal subtypes were found to be most significantly associated with NI, with activated stroma being enriched in neuro-invasive samples. We compared the results of differential expression and gene set enrichment analyses between NI and stromal subtypes from both bulk and single-cell RNA-seq data, which revealed a prominent role of myCAFs and common signatures between activated stroma and NI (e.g., ECM remodeling and TGF{beta} signaling) as well as signatures that changed in divergent directions for activated stroma and NI (e.g., iCAF markers). Our study presents support for the clinical implications of PDAC stromal subtypes by revealing their association with NI incidence, while providing biological insights into the interactions between diverse genetic programs that influence NI emergence.