Expression patterns of ligand genes originating from nontumoral cells. (A) Violin plots showing expression patterns of ligand genes in Fig.3 across anatomical layers. From left to right, samples from Patients 1 to 3 include mucosa, submucosa, muscularis propria, and subserosa/mesentery. For Patient 4, mucosa, submucosa, and muscularis propria. Y-axis, Log-normalized expression values. (B) Boxplots showing corresponding gene expression levels from GTEx datasets for external comparison.

Clinically relevant postoperative pancreatic fistula (CR-POPF) is the most consequential complication after distal pancreatectomy, occurring in up to one-third of cases. Video-based assessment (VBA) captures technical details not documented in operative reports. We conducted a VBA study to evaluate intraoperative factors associated with CR-POPF. We performed a retrospective single-center study of adults undergoing laparoscopic or robotic distal pancreatectomy (2019–2024). Three blinded reviewers coded predefined intraoperative variables from operative videos during the pancreatic transection. Associations with CR-POPF were assessed using Firth penalized logistic regression. Among 43 patients, 11 (25.6

PURPOSE:Small intestinal neuroendocrine tumors (SI-NET) frequently present as multifocal lesions, but the molecular mechanisms underlying their development and heterogeneity remain unclear. This study aimed to characterize the phenotypes of tumor cell populations across anatomic sites in patients with multifocal SI-NET and identify local microenvironmental factors influencing tumor development. EXPERIMENTAL DESIGN:Spatial transcriptomics was performed on 72 tissue microarray cores derived from 4 patients with multifocal SI-NETs that included tumoral and nontumoral tissues from various anatomic layers of the small intestine and regional metastatic sites. Unsupervised clustering, overrepresentation analysis, and ligand-receptor (L-R) pair analysis were used to define the tumor cell subtypes and associated signaling networks. External datasets were used for validation. Protein expression of selected genes was evaluated by immunohistochemistry and immunofluorescence. RESULTS:Unsupervised clustering revealed four major tumor cell subtypes: "mucosal," "mesenteric," "lymphatic," and "deep," based on their anatomic location and transcriptomic profiles. Each subtype exhibited distinct gene expression patterns and L-R interactions. The "mesenteric" and "lymphatic" subtypes exhibited distinct L-R pairs, such as NRG1-ERBB3 (HER3) and CXCL12-CXCR4, respectively. 5HT-HTR1D was found in all subtypes except "mucosal." Across the four subtypes, SST-SSTR1/2, PTN-NCL, MDK-NCL, and GJD2-GJD2 were consistently detected, suggesting fundamental roles in SI-NET biology. CONCLUSIONS:Although further validation is needed, our findings indicate that multifocal SI-NETs consist of spatially distinct tumor cell subtypes affected by local cellular interactions, providing insight into SI-NET intratumoral heterogeneity, possible microenvironment-triggered tumorigenesis, and potential subtype-targeted therapeutic strategies.

Inferred cellular compositions of individual spots used in the analysis. Bar plots on the left display the cellular composition of each subtype as determined by deconvolution analysis. Corresponding spots are shown on the image of the TMA slides, representing the regions selected for analysis. Pie charts within each spot illustrate the cellular composition within each spot. The color of each pie chart’s outline corresponds to the tumor subtype indicated in the legend on the right (m, mucosa; sm, submucosa; mp, muscularis propria; mes, mesenteric; tp, primary tumor; tm, tumor in mesentery; tl, tumor in lymph nodes).

Tumor gene module scores and thresholds for spot selection. The Y-axis represents the tumor gene module score. Red lines indicate the minimum threshold used to select tumoral spots, and blue lines maximum threshold for normal spots. m, mucosa; sm, submucosa; mp, muscularis propria; mes, mesenteric; tp, primary tumor; tm, tumor in mesentery; tl, tumor in lymph nodes.

Mucosal-like SI-NET cells in external datasets and heterogeneous serotonin signaling. (A) UMAP (left panels) and violin plots (right panels) showing NET cell subtypes with heterogeneous TPH1 expression in previously reported single-cell RNA-seq datasets (GSE292163) (11). Top panels, SiNET1 dataset; bottom panels, SiNET2 dataset. (B) Gene set enrichment analysis (GSEA) results of mucosal-like NET cells showing enrichment for gene sets characteristic of the ‘mucosal’ subtype. (C) KEGG pathway maps of serotonin-related signaling (hsa04726) comparing the ‘mucosal’ subtype in the current dataset with mucosal-like cells in the external dataset. Green indicates downregulated genes (log2 fold change), and red indicates upregulated genes.

BACKGROUND:The survival benefit of neoadjuvant chemotherapy (NAC) in early-stage pancreatic ductal adenocarcinoma (PDAC) remains uncertain. Although retrospective studies often suggest improved outcomes, these findings may be confounded by immortal time bias (ITB), the interval between diagnosis and treatment during which patients must survive to receive therapy, potentially inflating survival estimates. This study applied multiple bias-adjusted analytic methods to re-evaluate the association between NAC and overall survival in early-stage PDAC. METHODS:Using the National Cancer Database (2012-2017), we identified adults with resectable clinical T1 and T2 PDAC who underwent pancreatectomy. Overall survival was compared between multiagent NAC and upfront surgery by using Kaplan-Meier and multivariable Cox models. We used three statistical approaches to adjust for immortal time bias: 1) a 9-month landmark analysis; 2) a time-varying Cox regression model; and 3) a propensity-matched time-varying Cox model. RESULTS:Among 13,466 patients, 15.8% received NAC. Before bias adjustment, NAC was associated with longer median survival (33.4 vs. 25.7 months; HR 0.78, p < 0.001). However, after correcting for immortal time bias, this apparent survival advantage disappeared: landmark analysis (HR 0.94, p = 0.072), time-varying model (HR 1.06, p = 0.2), and matched cohort (HR 0.94, p = 0.2). CONCLUSIONS:This study shows that ITB significantly influences survival estimates in retrospective analyses of NAC for early-stage resectable PDAC. When appropriately adjusted, NAC was not associated with a survival advantage compared to upfront surgery. These findings underscore the need for methodological rigor in retrospective studies and caution against overinterpreting unadjusted survival advantages.

Immunofluorescence intensity correlation analysis. Scatter plots show background-corrected fluorescence intensities of serotonin, HTR1D, and pMAPK in tumor cells across tumor subtypes and in normal cells from the four patients (first and second rows) and the additional ten patients (third and fourth rows). Linear regression lines are overlaid, and the coefficients of determination (R²) and p-values in the manuscript were calculated using the lm() function from the R stats package with the formula y ∼ x.

ORA results of the cell subtypes. ORA was performed for each cell subtype, with C8: cell type signature gene sets downloaded from MSigDB (c8.all.v2023.2.Hs.symbols.gmt) as the input gene set. m, mucosa; sm, submucosa; mp, muscularis propria; mes, mesenteric; tp, primary tumor; tm, tumor in mesentery.

Images of 72 TMA cores used for analysis and corresponding original pathology images. Whole slide H&E image at low power with red circles indicating sampled sites of the cores and a zoomed H&E image of the corresponding TMA core (1 mm in diameter).

Scatter plot showing a negative correlation between the percentages of HTR1D-positive and Ki-67-positive tumor cells by immunohistochemistry. The coefficient of determination (R²) and P-value were calculated using the lm() function from the stats R package. A logarithmic model (formula: y ∼ log(x)) was applied due to the nonnormal distribution of the x-variable (Shapiro-Wilk test, p = 1.8 e-5).

Small intestine neuroendocrine tumors (SI-NETs) frequently present as multifocal primaries. We commonly observe microscopic lesions in the superficial layer of the small intestine of SI-NET patients. We aimed to define them as small intestinal neuroendocrine tumorlets (SINTs) and explore their clinical and biological significance. Twenty multifocal and twenty unifocal SI-NETs patients who received resection at a single institution were retrospectively reviewed. Four hundred and forty six archived pathological slides were examined for microscopic lesions located in the lamina propria, muscularis mucosa, and superficial submucosa. Clinicopathological associations and progression-free survival were analyzed. Previously published genomic data were re-analyzed. SINTs were identified in 50% of multifocal and 30% of unifocal SI-NET patients. Median SINT size was 95 μm, with a median distance of 2.2 mm from the nearest mass. Compared to the 'true unifocal' group (unifocal without SINT), the 'multifocal-spectrum' group (multifocal or unifocal with SINT) had higher BMI (median: 27.6 vs 22.8, P = 0.0060), higher rates of perineural invasion (OR: 5.5, P = 0.044), larger mesenteric mass (median: 2.6 vs 1.6 cm, P = 0.034), and more advanced pT stage (pT3 or pT4, OR: 7.2, P = 0.018). Genomic re-analysis suggested that 13% of cells in multifocal primary tumors could share clonal origins, possibly indicating clonal spread via SINTs. SINTs may serve as a new biomarker for multifocal spectrum with local aggressiveness. The actual frequency of multifocal SI-NET may be higher than currently recognized in clinical practice. Further studies are needed to validate their prognostic and biological significance.

Supplementary Table S1 Patient demographics for the spatial transcriptomics cohort. Supplementary Table S2 Tumor module genes. Supplementary Table S3 Number of analyzed spots per cell subtype. Supplementary Table S4 Lists of upregulated genes in each subtype. Supplementary Table S5 Gene expressions and results of Over-representation analysis by patient and tumor subtype. Supplementary Table S6 Ranked shared and significant ligand-receptor (L-R) pairs by tumor subtypes across patients with contribution values. Supplementary Table S7 NeuronChat analysis of serotonin-related ligand–receptor interactions. Supplementary Table S8 Summary of fluorescence intensity differences across tumor subtypes. Supplementary Table S9 Patient demographics for the additional immunofluprecence cohort. Supplementary Table S10 Demographics of patients with surgically resected SI-NET stratified by SSRI or SNRI use.

Representative images of serotonin-HTR1D-pMAPK triple immunofluorescence from an additional validation cohort of 10 patients. The first and second rows represent consecutive images derived from the same primary tumor of the same patient. Across all five rows, brightness and contrast channel settings (minimum, maximum, and gamma) were kept identical. Scale Bars, 400μm.

Grade progression of well-differentiated pancreatic neuroendocrine tumors (panNETs) can occur over time, with G1/2 to G3 being the most clinically relevant form. Here, we conducted a retrospective cohort study of 66 patients with initially G1/2 panNET (median initial Ki67, 4.6%). Patients were followed up for a median 6.8 years and had a median of two metachronous tumor biopsies over their disease course. 34.8% of patients underwent any form of grade progression, including G1 to G2/3 and G2 to G3, while 24.2% demonstrated G1/2 to G3 grade progression. Over a median 2.3 years, G1/2 to G3 grade progressors experienced a median Ki67 change of +27.0% (range, +6.4 to +48.7%). Subsequent biopsies showing progression to G3 had a median Ki67 value of 31.0% (range, 21.0-60.0%) and were more often performed following suspicious clinical behavior (75.0%) rather than routinely at the time of scheduled procedure/surgery (25.0%). Similar to prior studies, G1/2 to G3 grade progressors had worse overall survival from the time of metastatic disease (median, 4.8 years vs not reached for stably G1/2 disease; P = 0.002). Heavier pretreatment and heterogeneity or lack of uptake on somatostatin receptor imaging was independently associated with progression to G3. In the largest study of metachronous panNET biopsies to date, our findings show that baseline biopsies suggesting G1/2 disease may not accurately reflect future disease status, highlighting the possible limitations of using archived tissue to stratify patients into trials and/or choose future therapy. Additional work is needed to better understand the impact of prior therapies on grade progression and how to identify which lesions to best follow up for repeat biopsy.

(1) Background: Comprehensive evaluation of guideline-concordant care (GCC) across all PDAC stages has yet to be thoroughly conducted. This study aimed to characterize treatment patterns and assess factors associated with receiving GCC among patients with pancreatic ductal adenocarcinoma (PDAC) in California. (2) Methods: Data on adult patients with PDAC were extracted from the California Cancer Registry (2004-2020). GCC is defined according to the recommendations provided by the National Comprehensive Cancer Network. We used multivariable logistic regression to identify factors associated with receiving GCC. A Cox model was used to examine the association of GCC with overall survival. (3) Results: A total of 50,346 PDAC patients were included (stage 1: 10%; stage 2: 25%; stage 3: 11%; stage 4: 54%). Only 46.7% of all patients received GCC (stage 1: 20%; stage 2: 40%; stage 3: 69%; stage 4: 50%). Only 31% of stage 1 patients underwent surgery. Factors inversely associated with receiving GCC were Hispanic ethnicity (OR 0.78; p < 0.001), Black race (OR 0.74; p < 0.001), having no insurance (OR 0.40; p < 0.001]), and a Charlson-Deyo score of ≥2 (OR 0.68; p < 0.001). Adherence to GCC was associated with improved survival (Hazard Ratio 0.39; p < 0.001). Notably, patients with stage 1 PDAC who received GCC had a median survival of 47 months vs. 8 months for those who did not. (4) Conclusions: Although stage 1 PDAC patients have the greatest potential for survival with GCC, only 20% of patients received such treatment. Thus, it is crucial to identify and address the modifiable factors contributing to these suboptimal care patterns.