Intratumoral Cell Neighborhoods Coordinate Outcomes in Pancreatic Ductal Adenocarcinoma.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease characterized by a spatially heterogeneous tumor microenvironment. Within the PDA microenvironment, cells organize into communities where cell fate is influenced by neighboring cells of diverse ontogeny and function. However, it remains unclear how cell neighborhoods in the tumor microenvironment evolve with treatment and impact clinical outcomes. METHODS: Here, using automated chromogenic multiplex immunohistochemistry and unsupervised computational image analysis of human PDA tumors, we investigated cell neighborhoods in surgically resected tumors from patients with chemotherapy-naïve PDA (n = 59) and neoadjuvant chemotherapy-treated PDA (n = 57). Single cells were defined by lineage markers (CD3, CD8, Foxp3, CD68, CK19), proliferation (Ki67), and neighboring cells. RESULTS: Distinct intratumoral immune and tumor cell subsets were defined by neighboring cells. Higher content of stromal-associated macrophages was seen in chemotherapy-naïve tumors from long-term survivors (overall survival >3 years) compared with short-term survivors (overall survival <1 year), whereas immune-excluded tumor cells were higher in short-term survivors. Chemotherapy-treated vs -naïve tumors showed lower content of tumor-associated T cells and macrophages but similar densities of stromal-associated immune cells. However, proliferating tumor cell subsets with immune-rich neighborhoods were higher in chemotherapy-treated tumors. In a blinded analysis of tumors from patients treated with neoadjuvant chemotherapy, a composite index comprising lower quantities of immune-excluded tumor cells and higher spatially distinct immune cell subsets was associated with prolonged survival. CONCLUSIONS: Together, these data provide new insights into discrete cell communities in PDA and show their clinical relevance.

Integrated Molecular Characterization of Intraductal Papillary Mucinous Neoplasms: An NCI Cancer Moonshot Precancer Atlas Pilot Project.

Intraductal papillary mucinous neoplasms (IPMN) 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 multiregion sequencing for somatic mutations, there is no integrated assessment of molecular events, including copy-number alterations (CNA) and transcriptional changes that accompany IPMN progression. We performed laser capture microdissection on surgically resected IPMNs of varying grades of histologic dysplasia obtained from 23 patients, followed by whole-exome and whole-transcriptome sequencing. Overall, HG IPMNs displayed a significantly greater aneuploidy score than LG lesions, with chromosome 1q amplification being associated with HG progression and with cases that harbored co-occurring PDAC. Furthermore, the combined assessment of single-nucleotide variants (SNV) 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 as well as pathways related to glycosylation were a common feature of progression to HG. In addition, the established PDAC transcriptional subtypes (basal-like and classical) were readily apparent within IPMNs. 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 noninvasive precursor lesions, and reinforces that evolutionary pathways in IPMNs are heterogenous, comprised of both SNV and CNA-driven events. SIGNIFICANCE: Integrated molecular analysis of genomic and transcriptomic alterations in the multistep progression of 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.

Comparative Genomic Analysis of Pancreatic Acinar Cell Carcinoma (PACC) and Pancreatic Ductal Adenocarcinoma (PDAC) Unveils New Actionable Genomic Aberrations in PACC.

PURPOSE: Pure pancreatic acinar cell carcinomas (PACC) are rare malignancies with no established treatment. PACC demonstrates significant genetic intertumoral heterogeneity with multiple pathways involved, suggesting using targeted cancer therapeutics to treat this disease. We aggregated one of the largest datasets of pure PACC to examine the genomic variability and explore patient-specific therapeutic targets. EXPERIMENTAL DESIGN: PACC specimens (n = 51) underwent next-generation sequencing of DNA (n = 29) or whole exome (n = 22) and RNA (whole transcriptome, n = 29) at a commercial laboratory. We performed comparative analyses of a genomic cohort of pancreatic ductal adenocarcinomas (PDAC; n = 4,205). In parallel, we conducted a retrospective review of patients with PACC treated at Huntsman Cancer Institute (HCI). RESULTS: The real-world dataset included samples from 51 patients with PACC. We found key molecular differences between pure PACC and PDAC, highlighting the unique characteristics of pure PACC. Major differences in PACC include lower MAPK signaling and less stromal cell abundance compared with PDAC. Pure PACC showed genomic loss-of-heterozygosity to largely coincide with mutations in BRCA1, BRCA2, and PALB2. Of the 7 patients treated at HCI, one had a tumor that harbored a BRAF-V600E mutation. Leveraging precision oncology, this patient is being treated with encorafenib plus binimetinib, achieving an exceptionally durable and ongoing complete response of more than 3 years. CONCLUSIONS: There are major differences between PACC and PDAC, including downregulation of the MAPK signaling pathway, and less stromal cell abundance. In addition, genomic characterization of pure PACC revealed frequent targetable alterations, which can guide patient treatment.

Three pathologic criteria for reproducible diagnosis of colonic sessile serrated lesion versus hyperplastic polyp.

Colonic SSLs are thought to predispose to ∼30% of colonic adenocarcinomas. This increased risk, compared to benign HPs, makes their distinction vitally important. However, no gold standard exists to differentiate them, and wide observer variability is reported. To better distinguish these polyps, we investigated 94 serrated polyps (53 SSLs and 41 HPs) using an easy-to-apply pathologic scoring system that combines, for the first time, three established distinguishing features: polyp morphology, location, and size. As an additional novel approach, polyp size was assessed by serrated biopsy number compared to endoscopic size. RNA expression profiling served as an additional biomarker. The considerable morphologic overlap across serrated polyps was quantitated for the first time. Interobserver variability was assessed by 8 expert gastrointestinal pathologists. By ROC analysis, polyp size by biopsy number performed best, followed by polyp location and morphology (areas under the curves [AUCs] = 85.9%, 81.2%, and 65.9%, respectively). Optimal discrimination combined all 3 features (AUC = 92.9%). For polyp size, the biopsy number proved superior to endoscopic size (AUC = 85.9% versus 55.2%, P = .001). Interobserver variability analysis yielded the highest reported Fleiss and Kappa statistics (0.879) and percent agreement (96.8%), showing great promise toward improved diagnosis. The proposed 3-criteria pathologic system, combining size by biopsy number, location, and morphology, yields an improved, easy-to-use, and highly reproducible diagnostic approach for differentiating SSLs and HPs.

Quantitative Imaging Analysis Fluorescence In Situ Hybridization Validation for Clinical HER2 Testing in Breast Cancer.

CONTEXT.­: Quantitative imaging is a promising tool that is gaining wide use across several areas of pathology. Although there has been increasing adoption of morphologic and immunohistochemical analysis, the adoption of evaluation of fluorescence in situ hybridization (FISH) on formalin-fixed, paraffin-embedded tissue has been limited because of complexity and lack of practice guidelines. OBJECTIVE.­: To perform human epidermal growth factor receptor 2 (HER2) FISH validation in breast carcinoma in accordance with the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2018 guideline. DESIGN.­: Clinical validation of HER2 FISH was performed using the US Food and Drug Administration-approved dual-probe HER2 IQFISH (Dako, Carpinteria, California) with digital scanning performed on a PathFusion (Applied Spectral Imaging, Carlsbad, California) system. Validation parameters evaluated included z-stacking, classifier, accuracy, precision, software, and hardware settings. Finally, we evaluated the performance of digital enumeration on clinical samples in a real-world setting. RESULTS.­: The accuracy samples showed a final concordance of 95.3% to 100% across HER2 groups 1 to 5. During clinical implementation for HER2 groups 2, 3, and 4, we achieved a final concordance of 76% (95 of 125). Of these cases, only 8% (10 of 125) had discordances with clinical impact that could be identified algorithmically and triaged for manual review. CONCLUSIONS.­: Digital FISH enumeration is a useful tool to improve the efficacy of HER2 FISH enumeration and capture genetic heterogeneity across HER2 signals. Excluding cases with high background or poor image quality and manual review of cases with ASCO/CAP group discordances can further improve the efficiency of digital HER2 FISH enumeration.

Multianalyte Serum Biomarker Panel for Early Detection of Pancreatic Adenocarcinoma.

PURPOSE: We determined whether a large, multianalyte panel of circulating biomarkers can improve detection of early-stage pancreatic ductal adenocarcinoma (PDAC). MATERIALS AND METHODS: We defined a biologically relevant subspace of blood analytes on the basis of previous identification in premalignant lesions or early-stage PDAC and evaluated each in pilot studies. The 31 analytes that met minimum diagnostic accuracy were measured in serum of 837 subjects (461 healthy, 194 benign pancreatic disease, and 182 early-stage PDAC). We used machine learning to develop classification algorithms using the relationship between subjects on the basis of their changes across the predictors. Model performance was subsequently evaluated in an independent validation data set from 186 additional subjects. RESULTS: A classification model was trained on 669 subjects (358 healthy, 159 benign, and 152 early-stage PDAC). Model evaluation on a hold-out test set of 168 subjects (103 healthy, 35 benign, and 30 early-stage PDAC) yielded an area under the receiver operating characteristic curve (AUC) of 0.920 for classification of PDAC from non-PDAC (benign and healthy controls) and an AUC of 0.944 for PDAC versus healthy controls. The algorithm was then validated in 146 subsequent cases presenting with pancreatic disease (73 benign pancreatic disease and 73 early- and late-stage PDAC cases) and 40 healthy control subjects. The validation set yielded an AUC of 0.919 for classification of PDAC from non-PDAC and an AUC of 0.925 for PDAC versus healthy controls. CONCLUSION: Individually weak serum biomarkers can be combined into a strong classification algorithm to develop a blood test to identify patients who may benefit from further testing.

MYC-mediated resistance to trametinib and HCQ in PDAC is overcome by CDK4/6 and lysosomal inhibition.

Pharmacological inhibition of KRAS>RAF>MEK1/2>ERK1/2 signaling has provided no clinical benefit to patients with pancreatic ductal adenocarcinoma (PDAC). Interestingly, combined inhibition of MEK1/2 (with trametinib [T]) plus autophagy (with chloroquine [CQ] or hydroxychloroquine [HCQ]) demonstrated striking anti-tumor effects in preclinical models and in a patient (Patient 1). However, not all patients respond to the T/HCQ regimen, and Patient 1 eventually developed resistant disease. Here we report that primary or acquired resistance is associated with focal DNA copy number gains encompassing c-MYC. Furthermore, ectopic expression of c-MYC in PDAC cell lines rendered them T/HCQ resistant. Interestingly, a CDK4/6 inhibitor, palbociclib (P), also induced autophagy and overrode c-MYC-mediated T/HCQ resistance, such that P/HCQ promoted regression of T/HCQ-resistant PDAC tumors with elevated c-MYC expression. Finally, P/HCQ treatment of Patient 1 resulted in a biochemical disease response. These data suggest that elevated c-MYC expression is both a marker and a mediator of T/HCQ resistance, which may be overcome by the use of P/HCQ.

Distinguishing Gastrointestinal Leiomyomas From Muscularis Propria in Biopsy Specimens by Differential Expression of S100 Immunohistochemical Stain.

OBJECTIVES: Interpreting small biopsy specimens or fine-needle aspirations of gastrointestinal tract (GI) smooth muscle lesions may be challenging when the differential diagnosis includes leiomyoma vs muscularis propria (MP). We evaluated the utility of S100 staining in distinguishing GI leiomyomas from MP. METHODS: A search was conducted in our laboratory information system for cases of leiomyomas arising within the GI tract (2004-2021). Site-matched controls containing MP were selected (2018-2020). Five high-power fields (hpf) were counted on S100 immunohistochemical stains by two pathologists in the resections and by three different blinded pathologists in the biopsy specimens and analyzed. RESULTS: The median S100 count was 2.5/5 hpf in leiomyoma resection cases (n = 38), which was significantly lower than the median count of 548/5 hpf in MP (n = 19) with a P value of <.0001. The median S100 count in biopsy specimens (n = 16) was 1.2/5 hpf and within the expected range of 1 to 104/5 hpf (minimum-maximum value) established by the leiomyoma resections. S100 counts in the normal MP were significantly higher than those observed in leiomyomas (P < .001). CONCLUSIONS: S100 staining can aid in distinguishing a leiomyoma from MP in the GI tract, which is especially helpful when evaluating cases with limited sampling.

Diminished Immune Surveillance during Histologic Progression of Intraductal Papillary Mucinous Neoplasms Offers a Therapeutic Opportunity for Cancer Interception.

PURPOSE: Intraductal papillary mucinous neoplasms (IPMN) are bona fide precursors to pancreatic ductal adenocarcinoma (PDAC). While genomic alterations during multistep IPMN progression have been well cataloged, the accompanying changes within the tumor immune microenvironment (TIME) have not been comprehensively studied. Herein, we investigated TIME-related alterations during IPMN progression, using multiplex immunofluorescence (mIF) coupled with high-resolution image analyses. EXPERIMENTAL DESIGN: Two sets of formalin-fixed, paraffin-embedded tissue samples from surgically resected IPMNs were analyzed. The training set of 30 samples consisted of 11 low-grade IPMN (LG-IPMN), 17 high-grade IPMN (HG-IPMN), and 2 IPMN with PDAC, while a validation set of 93 samples comprised of 55 LG-IPMN and 38 HG-IPMN. The training set was analyzed with two panels of immuno-oncology-related biomarkers, while the validation set was analyzed with a subset of markers found significantly altered in the training set. RESULTS: Cell types indicative of enhanced immune surveillance, including cytotoxic and memory T cells, and antigen-experienced T cells and B cells, were all found at higher densities within isolated LG-IPMNs compared with HG-IPMNs. Notably, the TIME of LG-IPMNs that had progressed at the time of surgical resection (progressor LGD) resembled that of the synchronous HG-IPMNs, underscoring that attenuated immune surveillance occurs even in LG-IPMNs destined for progression. CONCLUSIONS: Our findings provide a basis for interception of cystic neoplasia to PDAC, through maintenance of sustained immune surveillance using vaccines and other prevention approaches.

Detection of circulating tumor DNA without a tumor-informed search using next-generation sequencing is a prognostic biomarker in pancreatic ductal adenocarcinoma.

The confounding effects of next-generation sequencing (NGS) noise on detection of low frequency circulating tumor DNA (ctDNA) without a priori knowledge of solid tumor mutations has limited the applications of circulating cell-free DNA (ccfDNA) in clinical oncology. Here, we use a 118 gene panel and leverage ccfDNA technical replicates to eliminate NGS-associated errors while also enhancing detection of ctDNA from pancreatic ductal adenocarcinomas (PDACs). Pre-operative ccfDNA and tumor DNA were acquired from 14 patients with PDAC (78.6% stage II-III). Post-operative ccfDNA was also collected from 11 of the patients within 100 days of surgery. ctDNA detection was restricted to variants corresponding to pathogenic mutations in PDAC present in both replicates. PDAC-associated pathogenic mutations were detected in pre-operative ccfDNA in four genes (KRAS, TP53, SMAD4, ALK) from five patients. Of the nine ctDNA variants detected (variant allele frequency: 0.08%-1.59%), five had a corresponding mutation in tumor DNA. Pre-operative detection of ctDNA was associated with shorter survival (312 vs. 826 days; χ2=5.4, P = 0.021). Guiding ctDNA detection in pre-operative ccfDNA based on mutations present in tumor DNA yielded a similar survival analysis. Detection of ctDNA in the post-operative ccfDNA with or without tumor-informed guidance was not associated with outcomes. Therefore, the detection of PDAC-derived ctDNA during a broad and untargeted survey of ccfDNA with NGS may be a valuable, non-invasive, prognostic biomarker to integrate into the clinical assessment and management of patients prior to surgery.

Differential methylation of G-protein coupled receptor signaling genes in gastrointestinal neuroendocrine tumors.

Neuroendocrine tumors (NETs) of the small intestine undergo large chromosomal and methylation changes. The objective of this study was to identify methylation differences in NETs and consider how the differentially methylated genes may impact patient survival. Genome-wide methylation and chromosomal copy number variation (CNV) of NETs from the small intestine and appendix were measured. Tumors were divided into three molecular subtypes according to CNV results: chromosome 18 loss (18LOH), Multiple CNV, and No CNV. Comparison of 18LOH tumors with MultiCNV and NoCNV tumors identified 901 differentially methylated genes. Genes from the G-protein coupled receptor (GPCR) pathways are statistically overrepresented in the differentially methylated genes. One of the highlighted genes from the GPCR pathway is somatostatin (SST), a clinical target for NETs. Patient survival based on low versus high methylation in all samples identified four significant genes (p < 0.05) OR2S2, SMILR, RNU6-653P, and AC010543.1. Within the 18LOH molecular subtype tumors, survival differences were identified in high versus low methylation of 24 genes. The most significant is TRHR (p < 0.01), a GPCR with multiple FDA-approved drugs. By separating NETs into different molecular subtypes based on chromosomal changes, we find that multiple GPCRs and their ligands appear to be regulated through methylation and correlated with survival. These results suggest opportunities for better treatment strategies for NETs based on molecular features.

Interobserver study on histologic features of idiopathic non-cirrhotic portal hypertension.

BACKGROUND: Histologic features of idiopathic non-cirrhotic portal hypertension (INCPH) may overlap with those without INCPH. Recently, these features have been recognized as part of the larger spectrum of porto-sinusoidal vascular disease (PSVD). We assessed interobserver agreement on histologic features that are commonly associated with INCPH and studied whether a provision of relevant clinical history improves interobserver agreement. METHODS: The examined histologic features include lobular (such as anisocytosis, nodular regeneration, sinusoidal dilatation, increased parenchymal draining veins, and incomplete fibrous septa) and portal tract changes (such as paraportal shunting vessel(s), portal tract remnant, increased number of portal vessels, and obliterative portal venopathy). Thirty-four archived liver samples from patients with (group A) and without (group B) INCPH were retrieved. A total of 90 representative images of lobules (L) and portal tracts (P) were distributed among 9 liver pathologists blinded to true clinical history. Each pathologist answered multiple choice questions based on the absence (Q1) or presence (Q2) of clinical history of portal hypertension. Fleiss' kappa coefficient analysis (unweighted) was performed to assess interobserver agreement on normal versus abnormal diagnosis, in L and P, based on Q1 and Q2. RESULTS: The kappa values regarding normal versus abnormal diagnosis were 0.24, 0.24, 0.18 and 0.18 for L-Q1, L-Q2, P-Q1, and P-Q2, respectively. With true clinical history provided, the kappa values were L- 0.32, P-0.17 for group A and L-0.12, P-0.14 for group B. Four pathologists changed their assessments based on the provided history. Interobserver agreement on the interpretation of L and P as normal versus abnormal was slight to fair regardless of provision of clinical history. CONCLUSIONS: Our findings indicate that the histologic features of INCPH/PSVD are not limited to patients with portal hypertension and are subject to significant interobserver variation.

The impact of squamous cell carcinoma histology on outcomes in nonmetastatic pancreatic cancer.

BACKGROUND: The prognosis for nonmetastatic, primary pancreatic squamous cell carcinoma (SCC) is thought to be poor compared with adenocarcinoma (AC); however, this is based on limited data. Additionally, the optimal definitive treatment strategy for nonmetastatic pancreatic SCC is unknown. METHODS: We analyzed patients with nonmetastatic pancreatic cancer using the National Cancer Database for patients diagnosed from 2006 to 2014. Patients were analyzed according to histology-only AC, adenosquamous carcinoma (A-SCC), and SCC were included. The primary endpoint was overall survival (OS) from the time of diagnosis. RESULTS: A total of 94 928 cases were included; 94 016 AC, 757 A-SCC, and 155 SCC. Median OS was lower for SCC (8.67 months), compared to AC (13.93 months) and A-SCC (12.71 months, P < .001). SCC was resected less often (25.5% vs 46.7% and 74.5%). On subgroup analysis of patients with pancreatic SCC, factors on multivariate analysis associated with improved survival included surgery (HR 0.19, P < .001), and chemotherapy (HR 0.22, P = .01). In 38 patients with SCC undergoing surgical resection, median OS improved (MS = 6.8 months without surgery vs 21.3 months with surgery, P < .001). CONCLUSIONS: Nonmetastatic pancreatic SCC presents with more advanced disease, which is less often surgically resected or treated with any definitive local therapy. In contrast, AC and A-SCC behave more similarly and have higher surgical resection rates and improved survival. In patients with nonmetastatic SCC of the pancreas, surgical resection provides the most significant survival benefit, with systemic chemotherapy providing a less significant benefit, and localized radiation providing no statistical benefit for any subgroup.

Publisher Correction: Protective autophagy elicited by RAF→MEK→ERK inhibition suggests a treatment strategy for RAS-driven cancers.

In the version of this article initially published, the label over the bottom schematic in Fig. 1a was "pH > 5.0"; it should have been "pH < 5.0". Further, the original article misspelt the surname of Katrin P. Guillen as "Gullien". These errors have been corrected in the print, PDF and HTML versions of the article.

Protective autophagy elicited by RAF→MEK→ERK inhibition suggests a treatment strategy for RAS-driven cancers.

Pancreatic ductal adenocarcinoma (PDA) was responsible for ~ 44,000 deaths in the United States in 2018 and is the epitome of a recalcitrant cancer driven by a pharmacologically intractable oncoprotein, KRAS1-4. Downstream of KRAS, the RAF→MEK→ERK signaling pathway plays a central role in pancreatic carcinogenesis5. However, paradoxically, inhibition of this pathway has provided no clinical benefit to patients with PDA6. Here we show that inhibition of KRAS→RAF→MEK→ERK signaling elicits autophagy, a process of cellular recycling that protects PDA cells from the cytotoxic effects of KRAS pathway inhibition. Mechanistically, inhibition of MEK1/2 leads to activation of the LKB1→AMPK→ULK1 signaling axis, a key regulator of autophagy. Furthermore, combined inhibition of MEK1/2 plus autophagy displays synergistic anti-proliferative effects against PDA cell lines in vitro and promotes regression of xenografted patient-derived PDA tumors in mice. The observed effect of combination trametinib plus chloroquine was not restricted to PDA as other tumors, including patient-derived xenografts (PDX) of NRAS-mutated melanoma and BRAF-mutated colorectal cancer displayed similar responses. Finally, treatment of a patient with PDA with the combination of trametinib plus hydroxychloroquine resulted in a partial, but nonetheless striking disease response. These data suggest that this combination therapy may represent a novel strategy to target RAS-driven cancers.

CMV Disease in IBD: Comparison of Diagnostic Tests and Correlation with Disease Outcome.

Background: Significance of cytomegalovirus (CMV) in inflammatory bowel disease (IBD) is unclear due to pathobiology, numerous CMV tests, and disparate treatment outcomes. Methods: Retrospective chart review was done on patients with positive qualitative CMV tissue polymerase chain reaction (PCR) from 2005-2013 at a tertiary referral hospital. Frequency of PCR+, hematoxylin and eosin staining(HE)+, histopathology and immunohistochemistry (IHC)+ was assessed. IHC was assessed on a sample of PCR- tissues. Surgery rates were correlated with CMV testing and treatment. Results: PCR was done on 310 samples from 180 patients. Thirty-seven samples were PCR+ (51.4% PCR+ only, 35.1% IHC/PCR+, 13.5% HE/IHC/PCR+). The H&E frequently failed to detect CMV identified on extensive IHC. Of 13 PCR- samples tested with IHC, 100% were negative. Twenty-five patients were CMV+ (40% PCR+, 40% IHC/PCR+, 20% HE/IHC/PCR+). Surgery rates increased with number of positive tests: 60% in IHC/PCR+ and 80% in HE/IHC/PCR+, compared to 26.8% in PCR- or PCR+ (P = 0.03, P = 0.02, respectively). There were 20/25 PCR+ patients who received CMV treatment. Surgery occurred in 80% of HE+ patients despite treatment and 100% of IHC+ patients without treatment. Conclusions: Rates of CMV+ testing and surgical risk varied by test modality. PCR+ results were most frequent but alone did not detect clinically significant CMV. HE+ testing was least frequent and associated with highest surgical rate, despite treatment. CMV treatment may benefit IHC+ patients most, supporting immunostaining as optimal diagnostic test for clinically significant CMV in IBD. In PCR+ samples, HE frequently did not detect CMV identified on extensive IHC. In PCR- samples, data suggest IHC is likely negative. Consider using qualitative PCR to guide extensive immunostaining.

Intramucosal lipomas of the colon implicate Cowden syndrome.

Intramucosal lipomas are rare and easily overlooked by pathologists, despite their diagnostic significance for Cowden syndrome (PTEN hamartoma tumor syndrome), an inherited multiorgan cancer syndrome. Only 25-35% of patients harbor identifiable PTEN mutations, thus clinical features, like intramucosal lipomas, remain the mainstay of diagnosis. The significance and diagnostic approach to intramucosal lipomas have not been thoroughly addressed in the literature. Intramucosal lipomas are mimicked by pseudolipomatosis coli, an artifactual mucosal gas infiltration from endoscopic insufflation. This differential was investigated by morphology and S-100 immunohistochemistry. Twenty-five colonic intramucosal lipomas were identified from 176 archival gastrointestinal lipomas from 1998 to 2017. Controls included 40 submucosal lipomas and 30 pseudolipomatoses. S-100 immunohistochemistry on all 95 lesions confirmed delicate fat vacuole membranous and nuclear S-100 staining in lipomas absent from pseudolipomatoses. Differentiating morphology between intramucosal lipoma and pseudolipomatosis, respectively, included consistently large, regular fat vacuoles (92% vs 7%), associated spindle cells (80% vs 0%), and mucosal lymphoid aggregate involvement (12% vs 80%). Of the 25 intramucosal lipomas, five patients (20%) had confirmed Cowden syndrome (four with PTEN mutations). In four of these Cowden patients, the intramucosal lipoma was the index diagnostic lesion. Three (12%) intramucosal lipoma patients had additional clinical features associated with Cowden syndrome, but did not meet the diagnostic criteria. Sporadic-type intramucosal lipomas were identified in 17 patients (68%) without evidence of Cowden syndrome, including three with normal PTEN genetic testing. No distinguishing endoscopic or pathologic polyp features were identified between sporadic and syndromic intramucosal lipomas. These data provide evidence that intramucosal lipomas are important harbingers of Cowden syndrome, making up approximately one-third of this series, the largest in the literature. We also show for the first time that two-thirds of intramucosal lipomas are sporadic. Gastrointestinal pathologists, gastroenterologists, and geneticists should increase their awareness of this subtle but diagnosable lesion strongly associated with Cowden syndrome.

PD-L1 protein expression in tumour cells and immune cells in mismatch repair protein-deficient and -proficient colorectal cancer: the foundation study using the SP142 antibody and whole section immunohistochemistry.

AIMS: Routine application of PD-L1 immunohistochemistry (IHC) in colorectal cancer (CRC) is limited due to lack of standardized scoring criteria, antibody clones, and intratumoral staining heterogeneity. We assessed PD-L1 protein expression on full face CRC tissue sections and applied two algorithms based on the published clinical trials that support the recent FDA approval for immune checkpoint inhibitors (ICPI) therapy in non-small cell lung cancer (NSCLC). METHODS: PD-L1/CD274 IHC (Roche/Ventana, clone SP142) was performed on representative tumour blocks from 52 mismatch repair-deficient (MMR-D) and 52 MMR-proficient (MMR-P) CRCs. Membranous PD-L1 expression was scored for the tumour cell (TC) and tumour-infiltrating immune cell (IC) components. PD-L1 positivity status was determined based on the published NSCLC clinical trials that utilized the Ventana SP142 assay. Hybrid capture-based comprehensive genomic profiling (CGP) was performed on a separate set of 2268 clinically advanced CRCs and the frequency of PD-L1/PD-L2 amplification was determined. RESULTS: PD-L1 expression in the TC and IC correlated with MMR-D (p=0.013, p<0.0001), T stage (p=0.036, p=0.0036) and clinical stage (p=0.022, p=0.0037). PD-L1 positivity status correlated with MMR-D by two algorithms. Five of 2268 (<1%) advansced CRCs demonstrated amplification of either the PD-L1 or PD-L2 genes by CGP. CONCLUSIONS: PD-L1 expression in TC and IC is associated with advanced stage and MMR-D. PD-L1 positivity status by the published algorithm is associated with MMR-D. PD-L1 amplification is extremely uncommon in CRC. Evaluation of whole tissue section and incorporation of IC staining enhance the sensitivity to screen patients who may benefit from ICPI therapy.

Chemoprevention with Cyclooxygenase and Epidermal Growth Factor Receptor Inhibitors in Familial Adenomatous Polyposis Patients: mRNA Signatures of Duodenal Neoplasia.

To identify gene expression biomarkers and pathways targeted by sulindac and erlotinib given in a chemoprevention trial with a significant decrease in duodenal polyp burden at 6 months (P < 0.001) in familial adenomatous polyposis (FAP) patients, we biopsied normal and polyp duodenal tissues from patients on drug versus placebo and analyzed the RNA expression. RNA sequencing was performed on biopsies from the duodenum of FAP patients obtained at baseline and 6-month endpoint endoscopy. Ten FAP patients on placebo and 10 on sulindac and erlotinib were selected for analysis. Purity of biopsied polyp tissue was calculated from RNA expression data. RNAs differentially expressed between endpoint polyp and paired baseline normal were determined for each group and mapped to biological pathways. Key genes in candidate pathways were further validated by quantitative RT-PCR. RNA expression analyses of endpoint polyp compared with paired baseline normal for patients on placebo and drug show that pathways activated in polyp growth and proliferation are blocked by this drug combination. Directly comparing polyp gene expression between patients on drug and placebo also identified innate immune response genes (IL12 and IFNγ) preferentially expressed in patients on drug. Gene expression analyses from tissue obtained at endpoint of the trial demonstrated inhibition of the cancer pathways COX2/PGE2, EGFR, and WNT. These findings provide molecular evidence that the drug combination of sulindac and erlotinib reached the intended tissue and was on target for the predicted pathways. Furthermore, activation of innate immune pathways from patients on drug may have contributed to polyp regression. Cancer Prev Res; 11(1); 4-15. ©2017 AACRSee related editorial by Shureiqi, p. 1.