Applications of Artificial Intelligence in Lung Pathology.

Artificial intelligence/machine learning tools are being created for use in pathology. Some examples related to lung pathology include acid-fast stain evaluation, programmed death ligand-1 (PDL-1) interpretation, evaluating histologic patterns of non-small-cell lung carcinoma, evaluating histologic features in mesothelioma associated with adverse outcomes, predicting response to anti-PDL-1 therapy from hematoxylin and eosin-stained slides, evaluation of tumor microenvironment, evaluating patterns of interstitial lung disease, nondestructive methods for tissue evaluation, and others. There are still some frameworks (regulatory, workflow, and payment) that need to be established for these tools to be integrated into pathology.

Genomic Staging of Multifocal Lung Squamous Cell Carcinomas Is Independent of the Comprehensive Morphologic Assessment.

INTRODUCTION: Morphologic and molecular data for staging of multifocal lung squamous cell carcinomas (LSCCs) are limited. In this study, whole exome sequencing (WES) was used as the gold standard to determine whether multifocal LSCC represented separate primary lung cancers (SPLCs) or intrapulmonary metastases (IPMs). Genomic profiles were compared with the comprehensive morphologic assessment. METHODS: WES was performed on 20 tumor pairs of multifocal LSCC and matched normal lymph nodes using the Illumina NovaSeq6000 S4-Xp (Illumina, San Diego, CA). WES clonal and subclonal analysis data were compared with histologic assessment by 16 thoracic pathologists. In addition, the immune gene profiling of the study cases was characterized by the HTG EdgeSeq Precision Immuno-Oncology Panel. RESULTS: By WES data, 11 cases were classified as SPLC and seven cases as IPM. Two cases were technically suboptimal. Analysis revealed marked genomic and immunogenic heterogeneity, but immune gene expression profiles highly correlated with mutation profiles. Tumors classified as IPM have a large number of shared mutations (ranging from 33.5% to 80.7%). The agreement between individual morphologic assessments for each case and WES was 58.3%. One case was unanimously interpreted morphologically as IPM and was in agreement with WES. In a further 17 cases, the number of pathologists whose morphologic interpretation was in agreement with WES ranged from two (one case) to 15 pathologists (one case) per case. Pathologists showed a fair interobserver agreement in the morphologic staging of multiple LSCCs, with an overall kappa of 0.232. CONCLUSIONS: Staging of multifocal LSCC based on morphologic assessment is unreliable. Comprehensive genomic analyses should be adopted for the staging of multifocal LSCC.

Tenofovir Douche as HIV Preexposure Prophylaxis for Receptive Anal Intercourse: Safety, Acceptability, Pharmacokinetics, and Pharmacodynamics (DREAM 01).

BACKGROUND: Despite highly effective HIV preexposure prophylaxis (PrEP) options, no options provide on-demand, nonsystemic, behaviorally congruent PrEP that many desire. A tenofovir-medicated rectal douche before receptive anal intercourse may provide this option. METHODS: Three tenofovir rectal douches-220 mg iso-osmolar product A, 660 mg iso-osmolar product B, and 660 mg hypo-osmolar product C-were studied in 21 HIV-negative men who have sex with men. We sampled blood and colorectal tissue to assess safety, acceptability, pharmacokinetics, and pharmacodynamics. RESULTS: The douches had high acceptability without toxicity. Median plasma tenofovir peak concentrations for all products were several-fold below trough concentrations associated with oral tenofovir disoproxil fumarate (TDF). Median colon tissue mucosal mononuclear cell (MMC) tenofovir-diphosphate concentrations exceeded target concentrations from 1 hour through 3 to 7 days after dosing. For 6-7 days after a single product C dose, MMC tenofovir-diphosphate exceeded concentrations expected with steady-state oral TDF 300 mg on-demand 2-1-1 dosing. Compared to predrug baseline, HIV replication after ex vivo colon tissue HIV challenge demonstrated a concentration-response relationship with 1.9 log10 maximal effect. CONCLUSIONS: All 3 tenofovir douches achieved tissue tenofovir-diphosphate concentrations and colorectal antiviral effect exceeding oral TDF and with lower systemic tenofovir. Tenofovir douches may provide a single-dose, on-demand, behaviorally congruent PrEP option, and warrant continued development. Clinical Trials Registration . NCT02750540.

Computational pathology in 2030: a Delphi study forecasting the role of AI in pathology within the next decade.

BACKGROUND: Artificial intelligence (AI) is rapidly fuelling a fundamental transformation in the practice of pathology. However, clinical integration remains challenging, with no AI algorithms to date in routine adoption within typical anatomic pathology (AP) laboratories. This survey gathered current expert perspectives and expectations regarding the role of AI in AP from those with first-hand computational pathology and AI experience. METHODS: Perspectives were solicited using the Delphi method from 24 subject matter experts between December 2020 and February 2021 regarding the anticipated role of AI in pathology by the year 2030. The study consisted of three consecutive rounds: 1) an open-ended, free response questionnaire generating a list of survey items; 2) a Likert-scale survey scored by experts and analysed for consensus; and 3) a repeat survey of items not reaching consensus to obtain further expert consensus. FINDINGS: Consensus opinions were reached on 141 of 180 survey items (78.3%). Experts agreed that AI would be routinely and impactfully used within AP laboratory and pathologist clinical workflows by 2030. High consensus was reached on 100 items across nine categories encompassing the impact of AI on (1) pathology key performance indicators (KPIs) and (2) the pathology workforce and specific tasks performed by (3) pathologists and (4) AP lab technicians, as well as (5) specific AI applications and their likelihood of routine use by 2030, (6) AI's role in integrated diagnostics, (7) pathology tasks likely to be fully automated using AI, and (8) regulatory/legal and (9) ethical aspects of AI integration in pathology. INTERPRETATION: This systematic consensus study details the expected short-to-mid-term impact of AI on pathology practice. These findings provide timely and relevant information regarding future care delivery in pathology and raise key practical, ethical, and legal challenges that must be addressed prior to AI's successful clinical implementation. FUNDING: No specific funding was provided for this study.

Interactive Multimedia Reporting Technical Considerations: HIMSS-SIIM Collaborative White Paper.

Despite technological advances in the analysis of digital images for medical consultations, many health information systems lack the ability to correlate textual descriptions of image findings linked to the actual images. Images and reports often reside in separate silos in the medical record throughout the process of image viewing, report authoring, and report consumption. Forward-thinking centers and early adopters have created interactive reports with multimedia elements and embedded hyperlinks in reports that connect the narrative text with the related source images and measurements. Most of these solutions rely on proprietary single-vendor systems for viewing and reporting in the absence of any encompassing industry standards to facilitate interoperability with the electronic health record (EHR) and other systems. International standards have enabled the digitization of image acquisition, storage, viewing, and structured reporting. These provide the foundation to discuss enhanced reporting. Lessons learned in the digital transformation of radiology and pathology can serve as a basis for interactive multimedia reporting (IMR) across image-centric medical specialties. This paper describes the standard-based infrastructure and communications to fulfill recently defined clinical requirements through a consensus from an international workgroup of multidisciplinary medical specialists, informaticists, and industry participants. These efforts have led toward the development of an Integrating the Healthcare Enterprise (IHE) profile that will serve as a foundation for interoperable interactive multimedia reporting.

Pembrolizumab plus azacitidine in patients with chemotherapy refractory metastatic colorectal cancer: a single-arm phase 2 trial and correlative biomarker analysis.

BACKGROUND: DNA mismatch repair proficient (pMMR) metastatic colorectal cancer (mCRC) is not responsive to pembrolizumab monotherapy. DNA methyltransferase inhibitors can promote antitumor immune responses. This clinical trial investigated whether concurrent treatment with azacitidine enhances the antitumor activity of pembrolizumab in mCRC. METHODS: We conducted a phase 2 single-arm trial evaluating activity and tolerability of pembrolizumab plus azacitidine in patients with chemotherapy-refractory mCRC (NCT02260440). Patients received pembrolizumab 200 mg IV on day 1 and azacitidine 100 mg SQ on days 1-5, every 3 weeks. A low fixed dose of azacitidine was chosen in order to reduce the possibility of a direct cytotoxic effect of the drug, since the main focus of this study was to investigate its potential immunomodulatory effect. The primary endpoint of this study was overall response rate (ORR) using RECIST v1.1., and secondary endpoints were progression-free survival (PFS) and overall survival (OS). Tumor tissue was collected pre- and on-treatment for correlative studies. RESULTS: Thirty chemotherapy-refractory patients received a median of three cycles of therapy. One patient achieved partial response (PR), and one patient had stable disease (SD) as best confirmed response. The ORR was 3%, median PFS was 1.9 months, and median OS was 6.3 months. The combination regimen was well-tolerated, and 96% of treatment-related adverse events (TRAEs) were grade 1/2. This trial was terminated prior to the accrual target of 40 patients due to lack of clinical efficacy. DNA methylation on-treatment as compared to pre-treatment decreased genome wide in 10 of 15 patients with paired biopsies and was significantly lower in gene promoter regions after treatment. These promoter demethylated genes represented a higher proportion of upregulated genes, including several immune gene sets, endogenous retroviral elements, and cancer-testis antigens. CD8+ TIL density trended higher on-treatment compared to pre-treatment. Higher CD8+ TIL density at baseline was associated with greater likelihood of benefit from treatment. On-treatment tumor demethylation correlated with the increases in tumor CD8+ TIL density. CONCLUSIONS: The combination of pembrolizumab and azacitidine is safe and tolerable with modest clinical activity in the treatment for chemotherapy-refractory mCRC. Correlative studies suggest that tumor DNA demethylation and immunomodulation occurs. An association between tumor DNA demethylation and tumor-immune modulation suggests immune modulation and may result from treatment with azacitidine. Trial registration ClinicalTrials.gov, NCT02260440. Registered 9 October 2014, https://clinicaltrials.gov/ct2/show/NCT02260440 .

SPaRTAN, a computational framework for linking cell-surface receptors to transcriptional regulators.

The identity and functions of specialized cell types are dependent on the complex interplay between signaling and transcriptional networks. Recently single-cell technologies have been developed that enable simultaneous quantitative analysis of cell-surface receptor expression with transcriptional states. To date, these datasets have not been used to systematically develop cell-context-specific maps of the interface between signaling and transcriptional regulators orchestrating cellular identity and function. We present SPaRTAN (Single-cell Proteomic and RNA based Transcription factor Activity Network), a computational method to link cell-surface receptors to transcription factors (TFs) by exploiting cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) datasets with cis-regulatory information. SPaRTAN is applied to immune cell types in the blood to predict the coupling of signaling receptors with cell context-specific TFs. Selected predictions are validated by prior knowledge and flow cytometry analyses. SPaRTAN is then used to predict the signaling coupled TF states of tumor infiltrating CD8+ T cells in malignant peritoneal and pleural mesotheliomas. SPaRTAN enhances the utility of CITE-seq datasets to uncover TF and cell-surface receptor relationships in diverse cellular states.

Combined histopathological risk score using TP53 protein expression, CD8+ T cell density and intratumoral budding is an independent predictor of neoadjuvant therapy response in rectal adenocarcinoma.

AIMS: Neoadjuvant therapy is the recommended treatment for locally advanced rectal adenocarcinoma; however, there remains significant variability in response to therapy. Tumour protein 53 (TP53) has been associated with therapy response and prognosis with conflicting data. Recently, we demonstrated that immune cell density and intratumoral budding (ITB) are predictive factors in rectal cancer. We investigated the predictive value of TP53 immunohistochemistry with CD8+ T cell density and ITB on pretreatment biopsies of rectal adenocarcinoma for response to neoadjuvant therapy. METHODS AND RESULTS: Pretreatment biopsies of rectal adenocarcinoma from 117 patients with neoadjuvant therapy were analysed for TP53 expression by immunohistochemistry, ITB, CD8+ T cell density and mismatch repair protein (MMR) status. Most rectal adenocarcinomas displayed aberrant TP53 expression (86 of 117, 74%). Compared to wild-type TP53, aberrant TP53 expression was associated with proficient MMR status (P = 0.003) and low CD8+ T cell density (P = 0.001). Aberrant TP53 was significantly associated with a partial to poor response to neoadjuvant therapy [odds ratio (OR) = 2.42, 95% confidence interval (CI) = 1.04-5.62, P = 0.04]. A combined histopathological risk score (HRS) was created using CD8+ T cell density, ITB and TP53 expression. Patients were separated into low (none to one factor) and high (two to three factors) HRS categories. In the multivariable model, patients with a high HRS were 3.25-fold more likely to have a partial or poor response to neoadjuvant therapy (95% CI = 1.48-7.11, P = 0.003). CONCLUSIONS: Our study demonstrates that aberrant TP53 expression, high ITB and low CD8+ T cell density in pretreatment biopsies can help predict response to neoadjuvant therapy. These biomarkers may be helpful in identifying patients at risk for therapy resistance.

Crohn's disease-associated ATG16L1 T300A genotype is associated with improved survival in gastric cancer.

BACKGROUND: A non-synonymous single nucleotide polymorphism of the ATG16L1 gene, T300A, is a major Crohn's disease (CD) susceptibility allele, and is known to be associated with increased apoptosis induction in the small intestinal crypt base in CD subjects and mouse models. We hypothesized that ATG16L1 T300A genotype also correlates with increased tumor apoptosis and therefore could lead to superior clinical outcome in cancer subjects. METHODS: T300A genotyping by Taqman assay was performed for gastric carcinoma subjects who underwent resection from two academic medical centers. Transcriptomic analysis was performed by RNA-seq on formalin-fixed paraffin-embedded cancerous tissue. Tumor apoptosis and autophagy were determined by cleaved caspase-3 and p62 immunohistochemistry, respectively. The subjects' genotypes were correlated with demographics, various histopathologic features, transcriptome, and clinical outcome. FINDINGS: Of the 220 genotyped subjects, 163 (74%) subjects carried the T300A allele(s), including 55 (25%) homozygous and 108 (49%) heterozygous subjects. The T300A/T300A subjects had superior overall survival than the other groups. Their tumors were associated with increased CD-like lymphoid aggregates and increased tumor apoptosis without concurrent increase in tumor mitosis or defective autophagy. Transcriptomic analysis showed upregulation of WNT/ß-catenin signaling and downregulation of PPAR, EGFR, and inflammatory chemokine pathways in tumors of T300A/T300A subjects. INTERPRETATION: Gastric carcinoma of subjects with the T300A/T300A genotype is associated with repressed EGFR and PPAR pathways, increased tumor apoptosis, and improved overall survival. Genotyping gastric cancer subjects may provide additional insight for clinical stratification.

Validated Indices for Histopathologic Activity Predict Development of Colorectal Neoplasia in Ulcerative Colitis.

BACKGROUND AND AIMS: To correlate histologic activity in surveillance colonoscopies with the development of colorectal neoplasia in ulcerative colitis [UC]. METHODS: Colorectal biopsies during surveillance [N = 764] from 52 UC patients with colorectal neoplasia were compared to 122 patients without neoplasia enrolled in a prospective natural history registry. All biopsies were scored using validated histologic scoring systems (Geboes score, Nancy histopathologic index [NHI], and Robarts histopathologic index [RHI]). Clinical, endoscopic, and histologic data were correlated with the development of colorectal neoplasia. RESULTS: In multivariable analysis, mean RHI (hazard ratio [HR] 1.07 for each 1-unit increase in RHI, 95% confidence interval [CI] 1.03-1.12, p = 0.002) and mean NHI [HR 1.89 for each 1-unit increase in NHI, 95% CI 1.34-2.67, p = 0.002] for the entire surveillance period were significantly associated with colorectal neoplasia development. Shorter surveillance interval and increasing age were associated with increased risk of neoplasia development whereas mean Mayo endoscopic score was not significant. To generate a clinically useful measure of neoplasia risk, mean histologic activity in the preceding 5 years before the study endpoint was correlated with neoplasia development. In the preceding 5 years of surveillance, a mean RHI ≥ 8 had a 7.53-fold increased risk [95% CI 2.56-12.16, p < 0.001] and mean NHI ≥ 1.9 had a 5.89-fold increased risk [95% CI 2.18-15.92, p < 0.001] of developing colorectal neoplasia. CONCLUSIONS: Persistent histologic activity during multiple surveillance episodes is an independent predictor of colorectal neoplasia. Mean RHI and mean NHI during a 5-year colonoscopic surveillance period can be used to assess risk for colorectal neoplasia and optimize UC surveillance.

Serum IgG4 Subclass Deficiency Defines a Distinct, Commonly Encountered, Severe Inflammatory Bowel Disease Subtype.

BACKGROUND: Immunoglobulin G subclass 4 (IgG4) is hypothesized to play an immunomodulatory role, downregulating humoral immune responses. The role of this anti-inflammatory molecule in inflammatory bowel disease (IBD) has not been fully characterized. We sought to define alterations in serum IgG4 in patients with IBD and their association with multiyear disease severity. METHODS: We analyzed metadata derived from curated electronic health records from consented patients with IBD prospectively followed at a tertiary center over a 10-year time period. Patients with IBD with IgG4 serum levels available formed the study population. Demographics and multiyear clinical data were collected and analyzed. We stratified patients with IBD with low, normal, or high serum IgG4 levels. RESULTS: We found IgG4 characterized in 1193 patients with IBD and low IgG4 levels in 233 patients (20%) and elevated IgG4 levels in 61 patients (5%). An IgG4 deficiency did not significantly correlate with other antibody deficiencies. In a multiple Poisson regression analysis, low IgG4 was associated with more years on biologic agents (P = 0.002) and steroids (P = 0.049) and more hospital admissions (P < 0.001), clinic visits (P = 0.010), outpatient antibiotic prescriptions (P < 0.001), and CD-related surgeries (P = 0.011) during the study period after controlling for certain confounders. Elevated IgG4 was only associated with primary sclerosing cholangitis (P = 0.011). A cohort of patients with IgG4-deficient severe IBD received intravenous Ig replacement therapy, which benefited and was continued in 10 out of 11 individuals. CONCLUSIONS: An IgG4 subclass deficiency, distinct from other antibody deficiencies, occurred commonly in a referral IBD population and was associated with multiple markers of disease severity. This is the first association of IgG4 subclass deficiency with an inflammatory disease process. Further work is needed to define the mechanistic role of IgG4 deficiency in this severe IBD subgroup.

Intratumoral budding and automated CD8-positive T-cell density in pretreatment biopsies can predict response to neoadjuvant therapy in rectal adenocarcinoma.

Tumor budding and CD8-positive (+) T-cells are recognized as prognostic factors in colorectal adenocarcinoma. We assessed CD8+ T-cell density and intratumoral budding in pretreatment rectal cancer biopsies to determine if they are predictive biomarkers for response to neoadjuvant therapy and survival. Pretreatment biopsies of locally advanced rectal adenocarcinoma from 117 patients were evaluated for CD8+ T-cell density using automated quantitative digital image analysis and for intratumoral budding and correlated with clinicopathological variables on postneoadjuvant surgical resection specimens, response to neoadjuvant therapy, and survival. Patients with high CD8+ T-cell density (≥157 per mm2) on biopsy were significantly more likely to exhibit complete/near complete response to neoadjuvant therapy (66% vs. 33%, p = 0.001) and low tumor stage (0 or I) on resection (62% vs. 30%, p = 0.001) compared with patients with low CD8+ T-cell density. High CD8+ T-cell density was an independent predictor of response to neoadjuvant therapy with a 2.63 higher likelihood of complete response (95% CI 1.04-6.65, p = 0.04) and a 3.66 higher likelihood of complete/near complete response (95% CI 1.60-8.38, p = 0.002). The presence of intratumoral budding on biopsy was significantly associated with a reduced likelihood of achieving complete/near complete response to neoadjuvant therapy (odds ratio 0.36, 95% CI 0.13-0.97, p = 0.048). Patients with intratumoral budding on biopsy had a significantly reduced disease-free survival compared with patients without intratumoral budding (5-year survival 39% vs 87%, p < 0.001). In the multivariable model, the presence of intratumoral budding on biopsy was associated with a 3.35-fold increased risk of tumor recurrence (95% CI 1.25-8.99, p = 0.02). In conclusion, CD8+ T-cell density and intratumoral budding in pretreatment biopsies of rectal adenocarcinoma are independent predictive biomarkers of response to neoadjuvant therapy and intratumoral budding associates with patient survival. These biomarkers may be helpful in selecting patients who will respond to neoadjuvant therapy and identifying patients at risk for recurrence.

Altered Expression of the Epithelial Mucin MUC1 Accompanies Endoscopic Recurrence of Postoperative Crohn's Disease.

BACKGROUND: MUC1-glycoprotein is expressed at low levels and in fully glycosylated form on epithelial cells. Inflammation causes MUC1 overexpression and hypoglycosylation. We hypothesized that overexpression of hypoglycosylated MUC1 would be found in postoperative Crohn's disease (CD) recurrence and could be considered an additional biomarker of recurrence severity. METHODS: We examined archived neo-terminal ileum biopsies from patients with prior ileocecal resection who had postoperative endoscopic assessment of CD recurrence and given a Rutgeerts ileal recurrence score. Consecutive tissue sections were stained using 2 different anti-MUC1 antibodies, HMPV that recognizes all forms of MUC1 and 4H5 that recognizes only inflammation-associated hypoglycosylated MUC1. RESULTS: A total of 71 postoperative CD patients were evaluated. There was significant increase in MUC1 expression of both glycosylated/normal (P<0.0001) and hypoglycosylated/abnormal (P<0.0001) forms in patients with severe endoscopic CD recurrence (i3+i4), ileal score i2, compared with patients in endoscopic remission (i0+i1). Results were similar regardless of anti-TNF-α use. Although MUC1 expression and Rutgeerts scores were in agreement when characterizing the majority of cases, there were a few exceptions where MUC1 expression was characteristic of more severe recurrence than implied by Rutgeerts score. CONCLUSIONS: MUC1 is overexpressed and hypoglycosylated in neo-terminal ileum tissue of patients with postoperative CD recurrence. Increased levels are associated with more severe endoscopic recurrence scores, and this is not influenced by anti-TNF-α use. Discrepancies found between Rutgeerts scores and MUC1 expression suggest that addition of MUC1 as a biomarker of severity of postoperative CD recurrence may improve categorization of recurrence status and consequently treatment decisions.

Whole-slide Imaging: Clinical Workflows and Primary Diagnosis.

Digital pathology has made great strides in recent years culminating with the approval to market devices from the Food and Drug Administration. The pathology community is now poised to begin using these systems for diagnostic purposes. This article will discuss the preparatory steps needed to implement digital pathology as well as some implementation styles that may be sufficient for a pathology department.

Super-resolution imaging reveals the evolution of higher-order chromatin folding in early carcinogenesis.

Genomic DNA is folded into a higher-order structure that regulates transcription and maintains genomic stability. Although progress has been made on understanding biochemical characteristics of epigenetic modifications in cancer, the in-situ higher-order folding of chromatin structure during malignant transformation remains largely unknown. Here, using optimized stochastic optical reconstruction microscopy (STORM) for pathological tissue (PathSTORM), we uncover a gradual decompaction and fragmentation of higher-order chromatin folding throughout all stages of carcinogenesis in multiple tumor types, and prior to tumor formation. Our integrated imaging, genomic, and transcriptomic analyses reveal functional consequences in enhanced transcription activities and impaired genomic stability. We also demonstrate the potential of imaging higher-order chromatin disruption to detect high-risk precursors that cannot be distinguished by conventional pathology. Taken together, our findings reveal gradual decompaction and fragmentation of higher-order chromatin structure as an enabling characteristic in early carcinogenesis to facilitate malignant transformation, which may improve cancer diagnosis, risk stratification, and prevention.

Automated Quantitation of CD8-positive T Cells Predicts Prognosis in Colonic Adenocarcinoma With Mucinous, Signet Ring Cell, or Medullary Differentiation Independent of Mismatch Repair Protein Status.

Despite their association with DNA mismatch repair (MMR) protein deficiency, colonic adenocarcinomas with mucinous, signet ring cell, or medullary differentiation have not been associated with improved survival compared with conventional adenocarcinomas in most studies. Recent studies indicate that increased T-cell infiltration in the tumor microenvironment has a favorable prognostic effect in colonic adenocarcinoma. However, the prognostic effect of tumor-associated T cells has not been evaluated in histologic subtypes of colonic adenocarcinoma. We evaluated CD8-positive T-cell density in 259 patients with colonic adenocarcinoma, including 113 patients with tumors demonstrating mucinous, signet ring cell, or medullary differentiation, using a validated automated quantitative digital image analysis platform and correlated CD8-positive T-cell density with histopathologic variables, MMR status, molecular alterations, and survival. CD8-positive T-cell densities were significantly higher for MMR protein-deficient tumors (P<0.001), BRAF V600E mutant tumors (P=0.004), and tumors with medullary differentiation (P<0.001) but did not correlate with mucinous or signet ring cell histology (P>0.05 for both). In the multivariable model of factors predicting disease-free survival, increased CD8-positive T-cell density was associated with improved survival both in the entire cohort (hazard ratio=0.34, 95% confidence interval, 0.15-0.75, P=0.008) and in an analysis of patients with tumors with mucinous, signet ring cell, or medullary differentiation (hazard ratio=0.06, 95% confidence interval, 0.01-0.54, P=0.01). The prognostic effect of CD8-positive T-cell density was independent of tumor stage, MMR status, KRAS mutation, and BRAF mutation. Venous invasion was the only other variable independently associated with survival in both the entire cohort and in patients with tumors with mucinous, signet ring cell, or medullary differentiation. In summary, our results indicate that the prognostic value of MMR protein deficiency is most likely attributed to increased tumor-associated CD8-positive T cells and that automated quantitative CD8 T-cell analysis is a better biomarker of patient survival, particularly in patients with tumors demonstrating mucinous, signet ring cell, or medullary differentiation.

Quantitative image analysis for CD8 score in lung small biopsies and cytology cell-blocks.

INTRODUCTION: Immunotherapy has shown promising results in non-small cell lung cancer (NSCLC), for which tumour-infiltrating cytotoxic (CD8+) T cells play a critical role. We investigated the utility of image analysis (IA) to quantify CD8+ T cells in a series of matched small biopsies and resections of NSCLC. METHODS: CD8 immunohistochemistry was performed on cell-blocks (CB), core needle biopsies (CNB) and corresponding resections from primary NSCLCs. Slides were digitised using an Aperio AT2 scanner (Leica) and annotated by whole slide image (WSI) or fields of view occupied by tissue spots (TS). Quantitative IA was performed with a customised Aperio algorithm (Leica). CD8 scores (number of T cells with 1-3+ staining/total area) were then compared. RESULTS: Forty-four cases with CB or CNB material and a corresponding resection were analysed. Average CD8 score was determined in CB (7.67 WSI, 77.67 TS) and/or CNB (47.35 WSI, 325.67 TS), and corresponding resections (190.35 WSI, 336.58 TS). CD8 score concordance was highest (78.6%) for CNBs using WSI annotation. Overall, small biopsies (CB or CNB) correlated with the resection in 71.4% cases using WSI and 63.3% cases using TS annotation. IA performed better for low CD8 scores. CONCLUSIONS: These findings show that CD8 density in NSCLC can be quantified by IA in small biopsies and cell blocks, achieving the best concordance using WSI scores. Discrepancies were attributed to values near the cut-off and background detection of staining. These data warrant future studies with more cases and follow-up data to further investigate the clinical utility of IA for CD8 analysis in NSCLC.

Disease Characteristics and Severity in Patients With Inflammatory Bowel Disease With Coexistent Diabetes Mellitus.

BACKGROUND: Given the rising prevalence of diabetes mellitus (DM) and the limited data on its effect on the course of inflammatory bowel disease (IBD), we characterized multiyear patterns of disease severity in a cohort of IBD patients with coexistent DM. METHODS: Data of consented IBD patients followed prospectively in a natural history registry at a tertiary center between 2009 and 2017 were analyzed. Patients with ≥3 years of clinical follow-up were included. Patients identified with a diagnosis of DM were compared with 400 consecutive IBD controls without a diagnosis of DM, no laboratory evidence of hyperglycemia, and no history of antihyperglycemic treatment. RESULTS: Out of 2810 IBD patients, 141 (5%) had DM (IBD DM; 44% ulcerative colitis, 56% Crohn's disease, 48.2% female). IBD DM had higher use of 5-aminosalicylic acid (5ASA) agents (P = 0.04), narcotics (P < 0.001), and antibiotics (P = 0.007) but not immunomodulators and/or biologics compared with IBD controls. When analyzing biomarkers of severity, IBD DM demonstrated higher frequencies of elevated C-reactive protein (CRP; P = 0.006), elevated erythrocyte sedimentation rate (ESR; P = 0.001), eosinophilia (P = 0.004), monocytosis (P = 0.02), and hypoalbuminemia (P = 0.001). IBD DM had worse quality of life (mean Short Inflammatory Bowel Disease Questionnaire; P < 0.001). IBD DM had increased health care utilization compared with controls (emergency room usage P = 0.008, hospitalizations P < 0.001, gastroenterology clinic visits P < 0.001, and median annual charges P < 0.001). Among IBD DM patients, the use of immunomodulators and/or biologics was not associated with further complications as measured by antibiotic use or hospitalizations. CONCLUSIONS: This study of a large IBD cohort suggests that DM in IBD may be associated with increased disease severity and that there may be room for increasing use of highly effective immunomodulator and/or biologic agents in this group.

Cross-talk between Colon Cells and Macrophages Increases ST6GALNAC1 and MUC1-sTn Expression in Ulcerative Colitis and Colitis-Associated Colon Cancer.

Patients with ulcerative colitis have an increased risk of developing colitis-associated colon cancer (CACC). Changes in glycosylation of the oncoprotein MUC1 commonly occur in chronic inflammation, including ulcerative colitis, and this abnormally glycosylated MUC1 promotes cancer development and progression. It is not known what causes changes in glycosylation of MUC1. Gene expression profiling of myeloid cells in inflamed and malignant colon tissues showed increased expression levels of inflammatory macrophage-associated cytokines compared with normal tissues. We analyzed the involvement of macrophage-associated cytokines in the induction of aberrant MUC1 glycoforms. A coculture system was used to examine the effects of M1 and M2 macrophages on glycosylation-related enzymes in colon cancer cells. M2-like macrophages induced the expression of the glycosyltransferase ST6GALNAC1, an enzyme that adds sialic acid to O-linked GalNAc residues, promoting the formation of tumor-associated sialyl-Tn (sTn) O-glycans. Immunostaining of ulcerative colitis and CACC tissue samples confirmed the elevated number of M2-like macrophages as well as high expression of ST6GALNAC1 and the altered MUC1-sTn glycoform on colon cells. Cytokine arrays and blocking antibody experiments indicated that the macrophage-dependent ST6GALNAC1 activation was mediated by IL13 and CCL17. We demonstrated that IL13 promoted phosphorylation of STAT6 to activate transcription of ST6GALNAC1. A computational model of signaling pathways was assembled and used to test IL13 inhibition as a possible therapy. Our findings revealed a novel cellular cross-talk between colon cells and macrophages within the inflamed and malignant colon that contributes to the pathogenesis of ulcerative colitis and CACC.See related Spotlight on p. 160.

Complete Resolution of Mucosal Neutrophils Associates With Improved Long-Term Clinical Outcomes of Patients With Ulcerative Colitis.

BACKGROUND & AIMS: We investigated correlations between histologic features of the colonic mucosa in patients with ulcerative colitis (UC) and clinical outcomes during a 3-year follow-up period. METHODS: We obtained baseline biopsies from all colorectal segments (n = 889) from 281 patients with UC enrolled in a prospective study at a single center from 2009 through 2013. Biopsies were assessed in a blinded manner using validated histologic scoring systems (the Geboes score, Nancy histopathologic index, and Robarts histopathologic index). Clinical, endoscopic, and histologic data were collected and tested for correlations with systemic corticosteroid use, hospitalization, and colectomy within 3 years of the index colonoscopy. RESULTS: We found histologic evidence of UC activity (Geboes score ≥ 2B.1) in biopsies from 182 patients (65%) and endoscopic evidence of UC activity in 149 patients (53%) (substantial agreement, κ = 0.60). Histologic features of UC activity were associated with increased rates of systemic corticosteroid use, colectomy, and hospitalization in the entire cohort (P < .05 for all) and associated with increased rates of systemic corticosteroid use in an analysis limited to patients in endoscopic remission (P < .001). In patients in endoscopic remission, only histologic activity was independently associated with use of systemic corticosteroids (multivariate odds ratio, 6.34; 95% CI, 2.20-18.28; P = .001). Similar results were seen when the entire cohort was analyzed. Compared with patients without histologic evidence of UC activity, patients with only a small number of mucosal neutrophils still had higher rates of systemic corticosteroid use (P < .001). CONCLUSIONS: Histologic evidence of UC activity, including small numbers of neutrophils in the colonic mucosa, is the only factor independently associated with use of systemic corticosteroids. Complete resolution of neutrophil-associated inflammation should be a target for treatment of UC.