Exploring the mysterious mucinous appendiceal neoplasm.

Mucinous neoplasms of the appendix comprise a group of diagnostically challenging lesions that have generated significant controversy and confusion throughout the years, given their potential for aggressive behavior despite very bland cytologic features. Numerous classification schemes have been proposed to characterize and stage these lesions, but confusion remains among pathologists, surgeons, and oncologists regarding diagnostic criteria, therapeutic implications, and overall prognosis. This review summaries the current recommended nomenclature, histologic characteristics of each entity, and helpful features to distinguish neoplasia from benign mimics.

Adenomatoid tumours of the gastrointestinal tract - a case-series and review of the literature.

AIMS: Adenomatoid tumours are mesothelial-derived benign neoplasms with a predilection for the genital tract. Extragenital sites are rare and can cause significant diagnostic challenges. Herein, we describe the clinicopathological features of a cohort of adenomatoid tumours involving the gastrointestinal tract and liver in order to more clearly characterise their histological findings and aid in diagnosis. METHODS AND RESULTS: The pathology databases at four institutions were searched for adenomatoid tumours involving the gastrointestinal tract or liver, yielding eight cases. Available clinicoradiological and follow-up data were collected from the medical records. Six tumours were incidentally discovered during imaging studies or at the time of surgical exploration for unrelated conditions; presenting symptoms were unknown in two patients. Histologically, the tumours were well-circumscribed, although focal ill-defined borders were present in four cases. No infiltration of adjacent structures was identified. Architectural heterogeneity was noted in five (63%) tumours; an adenoid pattern often predominated. The neoplastic cells were flattened to cuboidal with eosinophilic cytoplasm. Cytoplasmic vacuoles mimicking signet ring-like cells were present in five (63%) cases. Three (38%) cases showed involvement of the mesothelium with reactive mesothelial hyperplasia. Cytological atypia or increased mitotic activity was not identified. The surrounding stroma ranged from oedematous/myxoid to densely hyalinised. Immunohistochemistry confirmed mesothelial origin in all cases evaluated. No patients developed recurrence of disease. CONCLUSIONS: The current study evaluates the clinicopathological findings in a collective series of gastrointestinal and hepatic adenomatoid tumours, correlating with those described in individually reported cases. We highlight common histological features and emphasise variable findings that could mimic a malignant neoplasm.

Validation of a Circulating Tumor DNA-Based Next-Generation Sequencing Assay in a Cohort of Patients with Solid tumors: A Proposed Solution for Decentralized Plasma Testing.

BACKGROUND: Characterization of circulating tumor DNA (ctDNA) has been integrated into clinical practice. Although labs have standardized validation procedures to develop single locus tests, the efficacy of on-site plasma-based next-generation sequencing (NGS) assays still needs to be proved. MATERIALS AND METHODS: In this retrospective study, we profiled DNA from matched tissue and plasma samples from 75 patients with cancer. We applied an NGS test that detects clinically relevant alterations in 33 genes and microsatellite instability (MSI) to analyze plasma cell-free DNA (cfDNA). RESULTS: The concordance between alterations detected in both tissue and plasma samples was higher in patients with metastatic disease. The NGS test detected 77% of sequence alterations, amplifications, and fusions that were found in metastatic samples compared with 45% of those alterations found in the primary tumor samples (p = .00005). There was 87% agreement on MSI status between the NGS test and tumor tissue results. In three patients, MSI-high ctDNA correlated with response to immunotherapy. In addition, the NGS test revealed an FGFR2 amplification that was not detected in tumor tissue from a patient with metastatic gastric cancer, emphasizing the importance of profiling plasma samples in patients with advanced cancer. CONCLUSION: Our validation experience of a plasma-based NGS assay advances current knowledge about translating cfDNA testing into clinical practice and supports the application of plasma assays in the management of oncology patients with metastatic disease. With an in-house method that minimizes the need for invasive procedures, on-site cfDNA testing supplements tissue biopsy to guide precision therapy and is entitled to become a routine practice. IMPLICATIONS FOR PRACTICE: This study proposes a solution for decentralized liquid biopsy testing based on validation of a next-generation sequencing (NGS) test that detects four classes of genomic alterations in blood: sequence mutations (single nucleotide substitutions or insertions and deletions), fusions, amplifications, and microsatellite instability (MSI). Although there are reference labs that perform single-site comprehensive liquid biopsy testing, the targeted assay this study validated can be established locally in any lab with capacity to offer clinical molecular pathology assays. To the authors' knowledge, this is the first report that validates evaluating an on-site plasma-based NGS test that detects the MSI status along with common sequence alterations encountered in solid tumors.

Colorectal carcinoma screening: Established methods and emerging technology.

Colorectal carcinoma screening programs have shown success in lowering both the incidence and mortality rate of colorectal carcinoma at a population level, in part because this carcinoma is relatively slow growing and has an identifiable premalignant lesion. Still, many patients do not undergo the recommended screening for colorectal carcinoma, and of those who do, a subset may be over- or under-diagnosed by the currently available testing methods. The primary purpose of this article is to review the data regarding currently available colorectal cancer screening modalities, which include fecal occult blood testing, direct colonic visualization, and noninvasive imaging techniques. In addition, readers will be introduced to a variety of biomarkers that may serve as stand-alone or adjunct tests in the future. Finally, there is a brief discussion of the current epidemiologic considerations that public health officials must address as they create population screening guidelines. The data we provide as laboratory physicians and scientists are critical to the construction of appropriate recommendations that ultimately decrease the burden of disease from colorectal carcinoma.

Post-inflammatory mucosal hyperplasia and appendiceal diverticula simulate features of low-grade appendiceal mucinous neoplasms.

Post-inflammatory mucosal hyperplasia and appendiceal diverticulosis simulate mucinous neoplasms, causing diagnostic confusion. Distinction between neoplasia and its mimics is particularly important since many authorities now consider all appendiceal mucinous neoplasms to be potentially malignant. The purpose of this study was to identify clinicopathologic and molecular features that may distinguish appendiceal mucinous neoplasms from non-neoplastic mimics. We retrospectively identified 92 mucinous lesions confined to the right lower quadrant, including 55 non-neoplastic examples of mucosal hyperplasia and/or diverticulosis and 37 low-grade neoplasms. Presenting symptoms, radiographic findings, appendiceal diameter, appearances of the lamina propria, non-neoplastic crypts, and epithelium, as well as mural changes were recorded. Twenty non-neoplastic lesions were subjected to KRAS mutational testing. Non-neoplastic appendices were smaller (p < 0.05) and more likely to present with symptoms of appendicitis (p < 0.05) than neoplasms. While post-inflammatory mucosal hyperplasia and diverticula often showed goblet cell-rich epithelium, extruded mucin pools, and patchy mural alterations with fibrosis, they always contained non-neoplastic crypts lined by mixed epithelial cell types and separated by lamina propria with predominantly preserved wall architecture. On the other hand, mucinous neoplasms lacked normal crypts (p < 0.05) and showed decreased lamina propria (p < 0.05) with diffusely thickened muscularis mucosae and lymphoid atrophy. Six (30%) non-neoplastic lesions contained KRAS mutations, particularly those containing goblet cell-rich hyperplastic epithelium. We conclude that distinction between neoplastic and non-neoplastic mucinous appendiceal lesions requires recognition of key morphologic features; KRAS mutational testing is an unreliable biomarker that cannot be used to assess biologic risk or confirm a diagnosis of neoplasia.

Smooth muscle tumors of the gastrointestinal tract: an analysis of prognostic features in 407 cases.

Smooth muscle tumors represent the second most common mural mesenchymal neoplasm in the gastrointestinal tract, but established criteria for prognostic assessment of these tumors are lacking. A large cohort of surgically resected intramural gastrointestinal smooth muscle tumors from 31 institutions was analyzed to identify potential prognostic features. Pathologic features were assessed by expert gastrointestinal and/or soft tissue pathologists at each center. Immunohistochemical confirmation was required. A total of 407 cases from the esophagus (n = 97, 24%), stomach (n = 180, 44%), small bowel (n = 74, 18%), and colorectum (n = 56, 14%) were identified. Patients ranged in age from 19 to 92 years (mean 55 years), with a slight female predominance (57%). Mean tumor size was 5.4 cm, with the largest tumor measuring 29 cm. Disease progression following surgery, defined as local recurrence, metastasis, or disease-related death, occurred in 56 patients (14%). Colorectal tumors were most likely to progress, followed by small bowel and gastric tumors. None of the esophageal tumors in this series progressed. Receiver operator characteristic analysis identified optimal cutoffs of 9.8 cm and 3 mitoses/5 mm2 for discriminating between progressive and non-progressive tumors. Histologic features strongly associated with progression by univariate analysis included moderate-to-severe atypia, high cellularity, abnormal differentiation (defined as differentiation not closely resembling that of normal smooth muscle), tumor necrosis, mucosal ulceration, lamina propria involvement, and serosal involvement (P < 0.0001 for all features). Age, sex, and margin status were not significantly associated with progression (P = 0.23, 0.82, and 0.07, respectively). A risk assessment table was created based on tumor site, size, and mitotic count, and Kaplan-Meier plots of progression-free survival for each subgroup revealed progression-based tiers. Based on our findings, it appears that nonesophageal gastrointestinal smooth muscle tumors measuring >10 cm and/or showing ≥3 mitoses/5 mm2 may behave aggressively, and therefore close clinical follow-up is recommended in these cases.

The diagnosis of clinically significant oesophageal Candida infections: a reappraisal of clinicopathological findings.

AIMS: Distinguishing true oesophageal Candida infections from oral contaminants is a common diagnostic issue. Historically, histological features believed to indicate true infection included epithelial invasion by pseudohyphae and intraepithelial neutrophils. Whether or not these features correlate with endoscopic lesions, symptoms and response to therapy has never been tested in a large cohort. The aim of this study was to determine whether specific histological features correlate with clinical and endoscopic findings when Candida is found in oesophageal biopsies. METHODS AND RESULTS: We reviewed 271 biopsies in which Candida was detected. Cases were evaluated for the presence of desquamated epithelial cells, location/type of fungal forms, neutrophils, and ulceration. Medical records were reviewed for clinical history, endoscopic lesions, and response to antifungal therapy. Statistical analysis was used to determine whether any histological features significantly correlated with clinical variables. There were 120 males and 151 females with a mean age of 42 years. Fifty-nine per cent had symptoms referable to the oesophagus, particularly dysphagia (36%). Most (73%) patients had abnormal endoscopic findings, with plaques, ulcers, or macroscopic evidence of oesophagitis. Seventy-one per cent of patients with documented antifungal therapy showed symptomatic improvement. Overall, there was no statistically significant correlation between any histological feature and presenting symptoms, endoscopic findings, or response to therapy. Importantly, the lack of pseudohyphae, demonstrable invasion of intact epithelium or neutrophilic infiltrates did not exclude clinically significant infection. CONCLUSIONS: We conclude that detection of Candida in oesophageal biopsies is always potentially clinically significant. Treatment decisions should be made on the basis of an integration of clinical, endoscopic and histological findings.

Cytomegalovirus reactivation in inflammatory bowel disease: an uncommon occurrence related to corticosteroid dependence.

Cytomegalovirus promotes mucosal injury in patients with inflammatory bowel disease, historically affecting 10-25% of ulcerative colitis patients with refractory disease. Viral reactivation is likely related to long-term corticosteroid therapy, which is no longer central to maintenance of patients with inflammatory bowel disease. We hypothesize that viral detection rates have decreased in the modern era, reflecting widespread use of immunomodulatory agents to control inflammation. We performed this study to evaluate the relationships between medical regimens and cytomegalovirus detection rates among patients with inflammatory bowel disease. We searched our database for all patients with established inflammatory bowel disease and severe flares diagnosed from 2002 to 2017. Patients maintained with corticosteroid therapy were considered to be corticosteroid-dependent and those treated with other agents were classified as corticosteroid-independent, provided they had not received corticosteroids within 6 months of colonoscopy. Biopsy samples were reviewed for viral inclusions and subjected to cytomegalovirus immunohistochemistry, and rates of viral detection were compared between groups. There were 135 corticosteroid-dependent patients; most had ulcerative colitis flares occurring during the 2002-2009 period. Patients with ulcerative colitis and Crohn disease were equally represented in the corticosteroid-independent group (n = 133) and most were evaluated for disease flares during the 2010-2017 interval. Cytomegalovirus was detected in 13 (8%) cases; 9 (69%) were diagnosed from 2002 to 2009 and all were obtained from corticosteroid-dependent patients (p = < 0.001). We conclude that rates of cytomegalovirus-related enterocolitis are declining among inflammatory bowel disease patients, reflecting a shift away from corticosteroid-based maintenance therapy in favor of more effective agents that do not promote viral reactivation.

Ultrasonographic Nonalcoholic Fatty Pancreas Is Associated with Advanced Fibrosis in NAFLD: A Retrospective Analysis.

BACKGROUND: Nonalcoholic fatty pancreas disease (NAF-P) is strongly linked with nonalcoholic fatty liver disease (NAFLD), but its relationship with advanced liver disease is unknown. AIMS: This study investigated the association between NAF-P and both advanced fibrosis and nonalcoholic steatohepatitis (NASH). METHODS: This retrospective study evaluated adults with biopsy-proven NAFLD with a sonogram within 1 year of liver biopsy. NAF-P was diagnosed by comparing the echogenicity of the pancreas to the kidney and was graded by severity. The primary outcome was the effect of NAF-P on the presence of advanced fibrosis and NASH, while secondary outcomes included the association of extensive NAF-P (grade II/III). Propensity score matching for independent risk factors of advanced fibrosis (age, gender, body mass index, and diabetes) was performed. RESULTS: One hundred and four patients were included in the study and 91 (87.5%) had NAF-P. After propensity score matching, NAF-P was significantly associated with advanced fibrosis (OR 10.52, p < 0.001) but not NASH (p = 0.27). Extensive NAF-P was predictive of advanced fibrosis (OR 3.35, p = 0.006) and NASH (OR 5.37, p < 0.001). NAF-P had a negative predictive value (NPV) of 93% for advanced fibrosis. When matching for the NAFLD fibrosis score in addition to the variables above, both NAF-P (OR 5.36, p = 0.001) and extensive NAF-P (OR 5.38, p = 0.002) still significantly predicted advanced fibrosis. CONCLUSION: NAF-P is predictive of advanced fibrosis, even when controlling for independent predictors of advanced fibrosis and the NAFLD fibrosis score. NAF-P has an excellent NPV and is a safe, inexpensive finding that can rule out advanced fibrosis.

Assessing colorectal cancer mismatch repair status in the modern era: a survey of current practices and re-evaluation of the role of microsatellite instability testing.

Results of DNA mismatch repair testing are used to detect Lynch syndrome and have prognostic and therapeutic implications among patients with sporadic colorectal carcinomas. Immunohistochemistry for mismatch repair proteins (MLH1, PMS2, MSH2, MSH6) and PCR for microsatellite instability are two established methods for assessing mismatch repair function. Older literature suggested a discordance rate of approximately 5% between these assays, leading some institutions to perform dual testing on all cases. Although universal mismatch repair testing is now recommended by multiple professional organizations, none provide guidelines regarding preferred assays. We surveyed 96 academic and nonacademic institutions to assess Lynch syndrome screening practices and evaluated discordance rates between immunohistochemistry and PCR among 809 colorectal cancers tested in our own institution. Our survey demonstrated no significant differences between academic and nonacademic practices with respect to testing strategies. Eighty six percent performed universal screening, and usually (76%) employed immunohistochemistry on initial biopsy samples. Only 20% employed PCR; these were mostly academic practices that used both immunohistochemistry and PCR (p < 0.01 compared with the nonacademic groups). Loss of MLH1/PMS2 staining was often (90%) followed by either BRAF mutational analysis or MLH1 methylation assays. Only 24% adhered to WHO recommendations to assign histologic grade based on mismatch repair status. We found only 3 cases (0.4%) with discordant immunohistochemistry and PCR results in our own practice: 1 reflected decreased MSH-6 staining in a neoadjuvantly treated microsatellite stable tumor, 1 MLH1-deficient tumor showed diminished MLH1/PMS2 in the tumor compared with internal control, and 1 case reflected an error in the molecular laboratory. Overall, our results showed extremely low discordance between methods assessing mismatch repair status and would suggest immunohistochemistry as the preferred single screening test. PCR can be reserved for cases that show equivocal immunostaining patterns.

Findings in exudates can help distinguish benign gastric ulcers from ulcerated adenocarcinomas.

AIMS: Most gastric carcinomas develop in association with mucosal atrophy and hypochlorhydria, whereas benign peptic ulcers are acid-related. Given that acid sterilises the gastric contents, we hypothesised that ulcerated gastric cancers may be associated with increased numbers of luminal microorganisms as compared with peptic ulcers, and that this feature may represent a helpful diagnostic clue to the presence of malignancy. We performed this study to determine whether the features of luminal debris, including microorganisms, from ulcerated gastric cancers were significantly different from those of debris associated with benign ulcers. METHODS AND RESULTS: We retrospectively identified 50 ulcerated adenocarcinomas and 50 site-matched peptic ulcers. Luminal debris was evaluated for the nature of inflammation, necrosis, and the presence of mixed bacterial colonies or yeasts. Non-lesional mucosa was assessed for chronic gastritis, Helicobacter pylori, chemical gastropathy, and intestinal metaplasia. Patients in both groups were adults (mean age: 69 years and 62 years, respectively) with similar amounts of inflammation and cellular necrosis in biopsy material. However, 76% of ulcerated cancers harboured non-H. pylori bacterial colonies, as compared with only 22% of peptic ulcers (P < 0.01). Filamentous bacteria and fungi were highly specific for carcinoma (98% and P = 0.02 for both comparisons). Background intestinal metaplasia was more common among gastric cancers than among peptic ulcers (50% versus 26%, P = 0.02), whereas chemical gastropathy was more commonly associated with the latter (50% versus 10%, P < 0.01). CONCLUSION: Gastric cancers may be colonised by non-H. pylori microorganisms. Detection of numerous bacterial colonies, filamentous bacteria or fungi in biopsy material obtained from ulcerated gastric lesions should raise suspicion for underlying malignancy.

Histologic Manifestations of Gastrointestinal Adenovirus Infection After Stem Cell Transplant.

Adenovirus can cause severe disease in hematopoietic stem cell transplant (HSCT) patients. Histopathologic features of this infection in gastrointestinal biopsies and their distinction from graft-versus-host disease (GVHD) have been incompletely studied. We retrospectively identified patients with gastrointestinal adenovirus infection. H&E-stained sections were reviewed and the histologic features were recorded. The extent of immunostaining was determined using a semiquantitative scale and a maximum number of positive cells per high-power field. Information regarding the clinical course and endoscopic findings were obtained from the electronic medical records. The study group included 32 HSCT patients. Most (81%) presented with diarrhea and detectable virus in the serum. Twenty patients had multiorgan involvement in the gastrointestinal tract, mostly in the duodenum (62%) and colon (56%). Characteristic features included apoptotic epithelial cells with nuclear disarray (84%) and tufted aggregates of degenerating epithelial cells (69%), the latter of which was more commonly seen in the study population more than a control group of HSCT patients with GI involvement by GVHD. Viral inclusions were limited to the superficial epithelium in 59% of samples, and the density of viral inclusions within biopsies was variable (grade 1: 40%, grade 2: 38%, and grade 3: 22%). Following therapy, 10 patients (30%) improved and 14 (42%) had progressive disease. Patients with disease progression were often older (64 vs. 36 years, P =0.01) with higher serologic viral loads, prior history of GVHD, multifocal involvement, and increased number and density of immunoreactive nuclei. Adenovirus infection elicits a spectrum of histologic changes that can simulate or occur in combination with gastrointestinal GVHD. Patients with progressive disease are more likely to have high viral loads and more extensive infection of the gastrointestinal tract.

Utility of C4d Immunohistochemistry in the Diagnosis of Esophageal Pemphigus Vulgaris.

Aims. To assess the utility of C4d immunohistochemistry for esophageal pemphigus vulgaris. Methods and results. We searched for patients with a history of esophageal pemphigus vulgaris who had esophageal biopsies for routine hematoxylin and eosin (H&E) staining. A total of 8 biopsies from 7 patients were available. We also identified 18 non-pemphigus esophageal biopsies for controls. C4d immunohistochemistry was performed on each biopsy. Five of 6 (83%) biopsies with classic pemphigus vulgaris histologic findings were positive for intercellular staining at the basal layer. The negative biopsy was in a patient that had recently received high-dose corticosteroid treatment for a flare. Two biopsies with atypical histologic features for pemphigus vulgaris had negative C4d staining but positive direct immunofluorescence (DIF) studies. Various nonspecific C4d staining patterns were observed in the controls, but none showed the intercellular staining pattern that was observed in pemphigus vulgaris. Conclusions. Suprabasal clefting with acantholysis and "tombstone effect" are described histologic features of pemphigus vulgaris on H&E. However, procedural artifact may mimic these findings. Currently, the gold standard for pemphigus vulgaris is DIF, which is not always available because it cannot routinely be performed on formalin-fixed paraffin embedded tissue. Our study shows that C4d immunohistochemistry may be a useful adjunct in evaluating esophageal pemphigus vulgaris.

Cytologic Findings and Ancillary Tests Results of Sclerosing Pneumocytoma: Our Institutional Experience.

INTRODUCTION: Sclerosing pneumocytoma (SP) is a rare benign tumor and a potential diagnostic pitfall. Our aim was to review the cytologic features of our surgically diagnosed SP cases including the clinical, immunohistochemical and available molecular findings. MATERIALS AND METHODS: A computerized search from 2013 to 2020 for surgical cases with corresponding cytology specimens diagnosed as SP was performed. The clinical data, cytology, and surgical specimens were collated for analysis. RESULTS: Six cytology specimens were collected. All were female (mean age = 35). Three have incidental lung nodules and three with cough. Cytologic findings showed variable architectural pattern (papillary, solid, singly scattered, acinar/rosette-like) and cellular heterogeneity (surface, stromal, epithelioid, plasmacytoid cells). Atypia was identified in 4/6 cases. The original cytology diagnoses were negative = 1, SP = 2 and adenocarcinoma = 3. The latter diagnoses were amended to SP after review of the surgical specimens. The three false positive cases on review have cytologic features mimicking adenocarcinoma. Immunohistochemical stains showed tumor cells (surface and stromal) were positive for TTF-1, and EMA with only the surface cells positive for pancytokeratin and Napsin A. Though two cases sent for molecular testing were negative for AKT1 or CTNNB1 exon 3 mutation, our panel did not evaluate AKT1 exon 4. CONCLUSIONS: SP is a diagnostic pitfall with 50% initially misdiagnosed as adenocarcinoma. Integrating the clinical/radiologic findings, cytologic features, and performance of immunohistochemistry on cell block are helpful in avoiding misdiagnosis. Molecular testing for recurrent mutations, if present, could be helpful for diagnosis and possible therapy options. However, routinely used molecular testing may not always capture relevant molecular markers for SP.

Spectrum of Findings Seen in Patients With IDH1/2-Mutant Cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma-with a growing incidence rate and poor prognosis-is not an aggressive tumor that is not uncommon. Molecular profiling can reveal actionable aberrations in at least a third of the tumors. This is especially so in the case of intrahepatic cholangiocarcinoma (ICC), where mutations in the isocitrate dehydrogenase 1 and 2 genes (IDH1/2) make up 15%-20% of these tumors. IDH1/2 mutant ICC is a rare disease that has not been adequately reported. To expand the spectrum of findings seen in these patients, we present a single institution case series. METHODS AND RESULTS: We descriptively characterize the clinical, radiological, and histopathological findings of 12 such patients. IDH1/2 mutant ICC was found in elderly women, with two-thirds of patients having additional co-mutations. Anecdotally, patients who did receive systemic and/or locoregional therapies had long-term durable outcomes. CONCLUSION: Our findings indicate an increasing need to personalize an approach for these patients with specific molecular alterations. With the advent of the IDH1 inhibitor ivosidenib and other inhibitors in this space, IDH1/2 mutation has both prognostic and predictive value. Our series builds upon the patterns and findings seen in these patients.

Integrative Transcriptomic and Single-Cell Protein Characterization of Colorectal Carcinoma Delineates Distinct Tumor Immune Microenvironments Associated with Overall Survival.

Colorectal carcinoma (CRC) is a heterogeneous group of tumors with varying therapeutic response and prognosis, and evidence suggests the tumor immune microenvironment (TIME) plays a pivotal role. Using advanced molecular and spatial biology technologies, we aimed to evaluate the TIME in patients with CRC to determine whether specific alterations in the immune composition correlated with prognosis. We identified primary and metastatic tumor samples from 31 consented patients, which were profiled with whole-exome sequencing and bulk RNA-seq. Immune cell deconvolution followed by gene set enrichment analysis and unsupervised clustering was performed. A subset of tumors underwent in situ analysis of the TIME spatial composition at single-cell resolution through Imaging Mass Mass Cytometry. Gene set enrichment analysis revealed two distinct groups of advanced CRC, one with an immune activated phenotype and the other with a suppressed immune microenvironment. The activated TIME phenotype contained increased Th1 cells, activated dendritic cells, tertiary lymphoid structures, and higher counts of CD8+ T cells whereas the inactive or suppressed TIME contained increased macrophages and a higher M2/M1 ratio. Our findings were further supported by RNA-seq data analysis from the TCGA CRC database, in which unsupervised clustering also identified two separate groups. The immunosuppressed CRC TIME had a lower overall survival probability (HR 1.66, p=0.007). This study supports the pertinent role of the CRC immune microenvironment in tumor progression and patient prognosis. We characterized the immune cell composition to better understand the complexity and vital role that immune activity states of the TIME play in determining patient outcome.

Bcl-xL is translocated to the nucleus via CtBP2 to epigenetically promote metastasis.

Nuclear Bcl-xL is found to promote cancer metastasis independently of its mitochondria-based anti-apoptotic activity. How Bcl-xL is translocated into the nucleus and how nuclear Bcl-xL regulates histone H3 trimethyl Lys4 (H3K4me3) modification have yet to be understood. Here, we report that C-terminal Binding Protein 2 (CtBP2) binds to Bcl-xL via its N-terminus and translocates Bcl-xL into the nucleus. Knockdown of CtBP2 by shRNA decreases the nuclear portion of Bcl-xL and reverses Bcl-xL-induced invasion and metastasis in mouse models. Furthermore, knockout of CtBP2 not only reduces the nuclear portion of Bcl-xL but also suppresses Bcl-xL transcription. The binding between Bcl-xL and CtBP2 is required for their interaction with MLL1, a histone H3K4 methyltransferase. Pharmacologic inhibition of the MLL1 enzymatic activity reverses Bcl-xL-induced H3K4me3 and TGFß mRNA upregulation, as well as invasion. Moreover, the cleavage under targets and release using nuclease (CUT&RUN) assay coupled with next-generation sequencing reveals that H3K4me3 modifications are particularly enriched in the promotor regions of genes encoding TGFß and its signaling pathway members in cancer cells overexpressing Bcl-xL. Altogether, the metastatic function of Bcl-xL is mediated by its interaction with CtBP2 and MLL1 and this study offers new therapeutic strategies to treat Bcl-xL-overexpressing cancer.

NIPBL::NACC1 Fusion Hepatic Carcinoma.

Several reports describing a rare primary liver tumor with histologic features reminiscent of follicular thyroid neoplasms have been published under a variety of descriptive terms including thyroid-like, solid tubulocystic, and cholangioblastic cholangiocarcinoma. Although these tumors are considered to represent histologic variants, they lack classic features of cholangiocarcinoma and have unique characteristics, namely immunoreactivity for inhibin and NIPBL::NACC1 fusions. The purpose of this study is to present clinicopathologic and molecular data for a large series of these tumors to better understand their pathogenesis. We identified 11 hepatic tumors with these features. Immunohistochemical and NACC1 and NIPBL fluorescence in situ hybridization assays were performed on all cases. Four cases had available material for whole-genome sequencing (WGS) analysis. Most patients were adult women (mean age: 42 y) who presented with abdominal pain and large hepatic masses (mean size: 14 cm). Ten patients had no known liver disease. Of the patients with follow-up information, 3/9 (33%) pursued aggressive behavior. All tumors were composed of bland cuboidal cells with follicular and solid/trabecular growth patterns in various combinations, were immunoreactive for inhibin, showed albumin mRNA by in situ hybridization, and harbored the NIPBL::NACC1 fusion by fluorescence in situ hybridization. WGS corroborated the presence of the fusion in all 4 tested cases, high tumor mutational burden in 2 cases, and over 30 structural variants per case in 3 sequenced tumors. The cases lacked mutations typical of conventional intrahepatic cholangiocarcinoma. In this report, we describe the largest series of primary inhibin-positive hepatic neoplasms harboring a NIPBL::NACC1 fusion and the first WGS analysis of these tumors. We propose to name this neoplasm NIPBL:NACC1 fusion hepatic carcinoma.

Distinct clinical outcomes and biological features of specific KRAS mutants in human pancreatic cancer.

KRAS mutations in pancreatic ductal adenocarcinoma (PDAC) are suggested to vary in oncogenicity but the implications for human patients have not been explored in depth. We examined 1,360 consecutive PDAC patients undergoing surgical resection and find that KRASG12R mutations are enriched in early-stage (stage I) disease, owing not to smaller tumor size but increased node-negativity. KRASG12R tumors are associated with decreased distant recurrence and improved survival as compared to KRASG12D. To understand the biological underpinnings, we performed spatial profiling of 20 patients and bulk RNA-sequencing of 100 tumors, finding enhanced oncogenic signaling and epithelial-mesenchymal transition (EMT) in KRASG12D and increased nuclear factor κB (NF-κB) signaling in KRASG12R tumors. Orthogonal studies of mouse KrasG12R PDAC organoids show decreased migration and improved survival in orthotopic models. KRAS alterations in PDAC are thus associated with distinct presentation, clinical outcomes, and biological behavior, highlighting the prognostic value of mutational analysis and the importance of articulating mutation-specific PDAC biology.

Clinicopathologic and Molecular Features of Pancreatic Ductal Adenocarcinomas Harboring Alterations in COMPASS-like Complex Genes.

CONTEXT.­: Recent genome-wide sequencing studies have identified a subset of pancreatic ductal adenocarcinomas (PDACs) harboring significant alterations in epigenetic regulation genes, including the COMPASS-like complex genes. Whether this subset of PDACs has specific histologic characteristics or carries prognostic or therapeutic implications is unknown. OBJECTIVE.­: To determine the specific clinicopathologic and molecular features of PDACs carrying mutations in COMPASS-like complex genes. DESIGN.­: We analyzed a series of 103 primary and metastatic PDACs with comprehensive molecular profiling, including 13 PDACs carrying loss-of-function COMPASS-like complex gene alterations (study cohort). Another 45 patients carrying PDACs with wild-type COMPASS-like complex genes were used as the control group. RESULTS.­: PDACs within the study cohort were smaller, harboring frequent areas of poor differentiation and concurrent alterations in KRAS, TP53, SMAD4, and CDKN2A. A subset of metastatic PDACs from the study cohort showed squamous differentiation. There was a trend toward decreased survival in the study group. We further interrogated 2 public data sets and found that PDACs with COMPASS-like complex gene alterations have increased rates of TP53 mutation, body-tail location, poor differentiation or undifferentiated histology, and a higher death rate. CONCLUSIONS.­: COMPASS-like complex gene alterations likely represent a subset of more aggressive PDACs with poor or squamous differentiation histologically and increased concurrent TP53 mutations. These findings may have potential prognostic and therapeutic implications.