A molecular single-cell lung atlas of lethal COVID-19.

Respiratory failure is the leading cause of death in patients with severe SARS-CoV-2 infection1,2, but the host response at the lung tissue level is poorly understood. Here we performed single-nucleus RNA sequencing of about 116,000 nuclei from the lungs of nineteen individuals who died of COVID-19 and underwent rapid autopsy and seven control individuals. Integrated analyses identified substantial alterations in cellular composition, transcriptional cell states, and cell-to-cell interactions, thereby providing insight into the biology of lethal COVID-19. The lungs from individuals with COVID-19 were highly inflamed, with dense infiltration of aberrantly activated monocyte-derived macrophages and alveolar macrophages, but had impaired T cell responses. Monocyte/macrophage-derived interleukin-1ß and epithelial cell-derived interleukin-6 were unique features of SARS-CoV-2 infection compared to other viral and bacterial causes of pneumonia. Alveolar type 2 cells adopted an inflammation-associated transient progenitor cell state and failed to undergo full transition into alveolar type 1 cells, resulting in impaired lung regeneration. Furthermore, we identified expansion of recently described CTHRC1+ pathological fibroblasts3 contributing to rapidly ensuing pulmonary fibrosis in COVID-19. Inference of protein activity and ligand-receptor interactions identified putative drug targets to disrupt deleterious circuits. This atlas enables the dissection of lethal COVID-19, may inform our understanding of long-term complications of COVID-19 survivors, and provides an important resource for therapeutic development.

PD-1 Signaling Promotes Tumor-Infiltrating Myeloid-Derived Suppressor Cells and Gastric Tumorigenesis in Mice.

BACKGROUND & AIMS: Immune checkpoint inhibitors have limited efficacy in many tumors. We investigated mechanisms of tumor resistance to inhibitors of programmed cell death-1 (PDCD1, also called PD-1) in mice with gastric cancer, and the role of its ligand, PD-L1. METHODS: Gastrin-deficient mice were given N-methyl-N-nitrosourea (MNU) in drinking water along with Helicobacter felis to induce gastric tumor formation; we also performed studies with H/K-ATPase-hIL1B mice, which develop spontaneous gastric tumors at the antral-corpus junction and have parietal cells that constitutively secrete interleukin 1B. Mice were given injections of an antibody against PD-1 or an isotype control before tumors developed, or anti-PD-1 and 5-fluorouracil and oxaliplatin, or an antibody against lymphocyte antigen 6 complex locus G (also called Gr-1), which depletes myeloid-derived suppressor cells [MDSCs]), after tumors developed. We generated knock-in mice that express PD-L1 specifically in the gastric epithelium or myeloid lineage. RESULTS: When given to gastrin-deficient mice before tumors grew, anti-PD-1 significantly reduced tumor size and increased tumor infiltration by T cells. However, anti-PD-1 alone did not have significant effects on established tumors in these mice. Neither early nor late anti-PD-1 administration reduced tumor growth in the presence of MDSCs in H/K-ATPase-hIL-1ß mice. The combination of 5-fluorouracil and oxaliplatin reduced MDSCs, increased numbers of intra-tumor CD8+ T cells, and increased the response of tumors to anti-PD-1; however, this resulted in increased tumor expression of PD-L1. Expression of PD-L1 by tumor or immune cells increased gastric tumorigenesis in mice given MNU. Mice with gastric epithelial cells that expressed PD-L1 did not develop spontaneous tumors, but they developed more and larger tumors after administration of MNU and H felis, with accumulation of MDSCs. CONCLUSIONS: In mouse models of gastric cancer, 5-fluorouracil and oxaliplatin reduce numbers of MDSCs to increase the effects of anti-PD-1, which promotes tumor infiltration by CD8+ T cells. However, these chemotherapeutic agents also induce expression of PD-L1 by tumor cells. Expression of PD-L1 by gastric epithelial cells increases tumorigenesis in response to MNU and H felis, and accumulation of MDSCs, which promote tumor progression. The timing and site of PD-L1 expression is therefore important in gastric tumorigenesis and should be considered in design of therapeutic regimens.

Hepatic pathology in patients dying of COVID-19: a series of 40 cases including clinical, histologic, and virologic data.

The novel coronavirus SARS-CoV-2 (coronavirus disease 19, or COVID-19) primarily causes pulmonary injury, but has been implicated to cause hepatic injury, both by serum markers and histologic evaluation. The histologic pattern of injury has not been completely described. Studies quantifying viral load in the liver are lacking. Here we report the clinical and histologic findings related to the liver in 40 patients who died of complications of COVID-19. A subset of liver tissue blocks were subjected to polymerase chain reaction (PCR) for viral ribonucleic acid (RNA). Peak levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were elevated; median ALT peak 68 U/l (normal up to 46 U/l) and median AST peak 102 U/l (normal up to 37 U/l). Macrovesicular steatosis was the most common finding, involving 30 patients (75%). Mild lobular necroinflammation and portal inflammation were present in 20 cases each (50%). Vascular pathology, including sinusoidal microthrombi, was infrequent, seen in six cases (15%). PCR of liver tissue was positive in 11 of 20 patients tested (55%). In conclusion, we found patients dying of COVID-19 had biochemical evidence of hepatitis (of variable severity) and demonstrated histologic findings of macrovesicular steatosis and mild acute hepatitis (lobular necroinflammation) and mild portal inflammation. We also identified viral RNA in a sizeable subset of liver tissue samples.

Radiomics of MRI for pretreatment prediction of pathologic complete response, tumor regression grade, and neoadjuvant rectal score in patients with locally advanced rectal cancer undergoing neoadjuvant chemoradiation: an international multicenter study.

OBJECTIVE: To investigate whether pretreatment MRI-based radiomics of locally advanced rectal cancer (LARC) and/or the surrounding mesorectal compartment (MC) can predict pathologic complete response (pCR), neoadjuvant rectal (NAR) score, and tumor regression grade (TRG). METHODS: One hundred thirty-two consecutive patients with LARC who underwent neoadjuvant chemoradiation and total mesorectal excision (TME) were retrospectively collected from 2 centers in the USA and Italy. The primary tumor and surrounding MC were segmented on the best available T2-weighted sequence (axial, coronal, or sagittal). Three thousand one hundred ninety radiomic features were extracted using a python package. The most salient radiomic features as well as MRI parameter and clinical-based features were selected using recursive feature elimination. A logistic regression classifier was built to distinguish between any 2 binned categories in the considered endpoints: pCR, NAR, and TRG. Repeated k-fold validation was performed and AUCs calculated. RESULTS: There were 24, 87, and 21 T4, T3, and T2 LARCs, respectively (median age 63 years, 32 to 86). For NAR and TRG, the best classification performance was obtained using both the tumor and MC segmentations. The AUCs for classifying NAR 0 versus 2, pCR, and TRG 0/1 versus 2/3 were 0.66 (95% CI, 0.60-0.71), 0.80 (95% CI, 0.74-0.85), and 0.80 (95% CI, 0.77-0.82), respectively. CONCLUSION: Radiomics of pretreatment MRIs can predict pCR, TRG, and NAR score in patients with LARC undergoing neoadjuvant treatment and TME with moderate accuracy despite extremely heterogenous image data. Both the tumor and MC contain important prognostic information. KEY POINTS: • Machine learning of rectal cancer on images from the pretreatment MRI can predict important patient outcomes with moderate accuracy. • The tumor and the tissue around it both contain important prognostic information.

High-resolution genomic alterations in Barrett's metaplasia of patients who progress to esophageal dysplasia and adenocarcinoma.

The main risk factor for esophageal dysplasia and adenocarcinoma (DAC) is Barrett's esophagus (BE), characterized by intestinal metaplasia. The critical genomic mechanisms that lead to progression of nondysplastic BE to DAC remain poorly understood and require analyses of longitudinal patient cohorts and high-resolution assays. We tested BE tissues from 74 patients, including 42 nonprogressors from two separate groups of 21 patients each and 32 progressors (16 in a longitudinal cohort before DAC/preprogression-BE and 16 with temporally concurrent but spatially separate DAC/concurrent-BE). We interrogated genome-wide somatic copy number alterations (SCNAs) at the exon level with high-resolution SNP arrays in DNA from formalin-fixed samples histologically confirmed as nondysplastic BE. The most frequent abnormalities were SCNAs involving FHIT exon 5, CDKN2A/B or both in 88% longitudinal BE progressors to DAC vs. 24% in both nonprogressor groups (p = 0.0004). Deletions in other genomic regions were found in 56% of preprogression-BE but only in one nonprogressor-BE (p = 0.0004). SCNAs involving FHIT exon 5 and CDKN2A/B were also frequently detected in BE temporally concurrent with DAC. TP53 losses were detected in concurrent-BE but not earlier in preprogression-BE tissues of patients who developed DAC. CDKN2A/p16 immunohistochemistry showed significant loss of expression in BE of progressors vs. nonprogressors, supporting the genomic data. Our data suggest a role for CDKN2A/B and FHIT in early progression of BE to dysplasia and adenocarcinoma that warrants future mechanistic research. Alterations in CDKN2A/B and FHIT by high-resolution assays may serve as biomarkers of increased risk of progression to DAC when detected in BE tissues.

Low Frequency of Lymph Node Metastases in Patients in the United States With Early-stage Gastric Cancers That Fulfill Japanese Endoscopic Resection Criteria.

BACKGROUND & AIMS: In the West, early gastric cancer is increasingly managed with endoscopic resection (ER). This is, however, based on the assumption that the low prevalence and risk of lymph node metastases observed in Asian patients is applicable to patients in the United States. We sought to evaluate the frequency of and factors associated with metastasis of early gastric cancers to lymph nodes, and whether the Japanese ER criteria are applicable to patients in the US. METHODS: We performed a retrospective study of 176 patients (mean age 68.5 years; 59.1% male; 58.5% white) who underwent surgical resection with lymph node dissection of T1 and Tis gastric adenocarcinomas, staged by pathologists, at 7 tertiary care centers in the US from January 1, 1999, through December 31, 2016. The frequency of lymph node metastases and associated risk factors were determined. RESULTS: The mean size of gastric adenocarcinomas was 23.0 ± 16.6 mm-most were located in the lower-third of the stomach (67.0%), invading the submucosa (55.1%), and moderately differentiated (31.3%). Lymphovascular invasion was observed in 18.2% of lesions. Overall, 20.5% of patients had lymph node metastases. Submucosal invasion (odds ratio, 3.9; 95% CI, 1.4-10.7) and lymphovascular invasion (odds ratio, 4.6; 95% CI, 1.8-12.0) were independently associated with increased risk of metastasis to lymph nodes. The frequency of lymph node metastases among patients fulfilling standard and expanded Japanese criteria for ER were 0 and 7.5%, respectively. CONCLUSIONS: The frequency of lymph node metastases among patients with early gastric cancer in a US population is higher than that of published Asian series. However, early gastric cancer lesions that meet the Japanese standard criteria for ER are associated with negligible risk of metastasis to lymph nodes, so ER can be recommended for definitive therapy. Expanded criteria cancers appear to have a higher risk of metastasis to lymph nodes, so ER may be considered for select cases.

CDKN2A-p16 Deletion and Activated KRASG12D Drive Barrett's-Like Gland Hyperplasia-Metaplasia and Synergize in the Development of Dysplasia Precancer Lesions.

BACKGROUND & AIMS: Barrett's esophagus is the precursor of esophageal dysplasia and esophageal adenocarcinoma. CDKN2A-p16 deletions were reported in 34%-74% of patients with Barrett's esophagus who progressed to dysplasia and esophageal adenocarcinoma, suggesting that p16 loss may drive neoplastic progression. KRAS activation frequently occurs in esophageal adenocarcinoma and precancer lesions. LGR5+ stem cells in the squamocolumnar-junction (SCJ) of mouse stomach contribute as Barrett's esophagus progenitors. We aimed to determine the functional effects of p16 loss and KRAS activation in Barrett's-like metaplasia and dysplasia development. METHODS: We established mouse models with conditional knockout of CDKN2A-p16 (p16KO) and/or activated KRASG12D expression targeting SCJ LGR5+ cells in interleukin 1b transgenic mice and characterized histologic alterations (mucous-gland hyperplasia/metaplasia, inflammation, and dysplasia) in mouse SCJ. Gene expression was determined by microarray, RNA sequencing, and immunohistochemistry of SCJ tissues and cultured 3-dimensional organoids. RESULTS: p16KO mice exhibited increased mucous-gland hyperplasia/metaplasia versus control mice (P = .0051). Combined p16KO+KRASG12D resulted in more frequent dysplasia and higher dysplasia scores (P = .0036), with 82% of p16KO+KRASG12D mice developing high-grade dysplasia. SCJ transcriptome analysis showed several activated pathways in p16KO versus control mice (apoptosis, tumor necrosis factor-α/nuclear factor-kB, proteasome degradation, p53 signaling, MAPK, KRAS, and G1-to-S transition). CONCLUSIONS: p16 deletion in LGR5+ cell precursors triggers increased SCJ mucous-gland hyperplasia/metaplasia. KRASG12D synergizes with p16 deletion resulting in higher grades of SCJ glandular dysplasia, mimicking Barrett's high-grade dysplasia. These genetically modified mouse models establish a functional role of p16 and activated KRAS in the progression of Barrett's-like lesions to dysplasia in mice, representing an in vivo model of esophageal adenocarcinoma precancer. Derived 3-dimensional organoid models further provide in vitro modeling opportunities of esophageal precancer stages.

Erythrophagocytosis is not a reproducible finding in liver biopsies, and is not associated with clinical diagnosis of hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a rare disease with high mortality. Liver involvement is common (based on elevated liver function tests) with most patients demonstrating acute hepatitis. Liver biopsies are frequently obtained in the setting of suspected HLH for the purpose of identification of erythrophagocytosis, and if present, this finding is thought to suggest or support the diagnosis of HLH. However, there are problems with this approach; in particular, we do not know whether this finding is reproducible or whether it is specific to HLH. Therefore, we conducted a multi-institutional study in which experienced liver pathologists reviewed images taken from liver biopsies from patients with normal liver, acute hepatitis, possible HLH, and clinical HLH to determine if there was agreement about the presence or absence of erythrophagocytosis, and to ascertain whether the finding corresponds to a clinical diagnosis of HLH. Twelve liver pathologists reviewed 141 images in isolation (i.e., no clinical information or diagnosis provided). These came from 32 patients (five normal, 17 acute hepatitis, six HLH, four possible HLH). The pathologists classified each image as negative, equivocal, or positive for erythrophagocytosis. Kappa was .08 (no agreement) for case-level and 0.1 for image-level (1.4% agreement, based on two images which were universally considered negative). There was no difference in the proportion of pathologists who diagnosed erythrophagocytosis among those with different diagnoses at case or image-level (p = 0.82 and p = 0.82, respectively). Thus, erythrophagocytosis is an entirely unreliable histologic parameter in liver, as it is irreproducible and not demonstrably associated with a clinical disease (namely, HLH). Unless and until more reliable guidelines can be established, pathologists should refrain from commenting on the presence or absence of erythrophagocytosis in liver biopsy.

Schimmelpenning-Feuerstein-Mims syndrome with orbital choristoma and KRAS mutation: a current review and novel case report.

INTRODUCTION: Schimmelpenning-Feurstein-Mims Syndrome (SFMS) is a rare neurocutaneous disorder. Herein, we describe a novel case and review the phenotypic spectrum and molecular findings of SFMS from an ophthalmology perspective. METHODS: Clinical case including presentation, management, pathology, and genetic analysis is described. A literature search on Schimmelpenning-Feuerstein-Mims and its synonyms, Linear nevus sebaceous syndrome, Organoid nevus syndrome, Jadassohn nevus phacomatosis, and Solomon syndrome, was conducted. An updated review and description of published cases with identified genetic mutations are described. RESULTS: A 13-year-old boy with SFMS presented with acute right eye pain and an enlarging orbital mass. Excisional biopsy of the mass revealed an orbital choristoma. Genetic analysis of the orbital tumor confirmed a KRAS c.35 G>A, p.G12D mutation. A literature search revealed 19 cases of SFMS with mutations in the RAS-pathway. KRAS, HRAS, and NRAS mutations were identified in 74%, 21%, and 5% of patients, respectively. Ophthalmic pathology was seen in 83% of patients. Systemic findings varied and involved the skin, central nervous system, and eyes most commonly. DISCUSSION: SFMS, a rare neurocutaneous disorder, results from postzygotic mosaic mutations in the RAS/MAPK pathway. Patients present with various systemic findings and ophthalmic manifestations occur in most cases. This is the first case description of a KRAS mutation identified in an orbital choristoma in SFMS. The disease is described under various names in the literature, and we propose that all syndromic cases with mosaic RAS mutations be reported under the eponym, SFMS.

Chemotherapy and Immune Checkpoint Blockade for Gastric and Gastroesophageal Junction Adenocarcinoma.

Importance: Combining immune checkpoint blockade (ICB) with chemotherapy improves outcomes in patients with metastatic gastric and gastroesophageal junction (G/GEJ) adenocarcinoma; however, whether this combination has activity in the perioperative setting remains unknown. Objective: To evaluate the safety and preliminary activity of perioperative chemotherapy and ICB followed by maintenance ICB in resectable G/GEJ adenocarcinoma. Design, Setting, and Participants: This investigator-initiated, multicenter, open-label, single-stage, phase 2 nonrandomized controlled trial screened 49 patients and enrolled 36 patients with resectable G/GEJ adenocarcinoma from February 10, 2017, to June 17, 2021, with a median (range) follow-up of 35.2 (17.4-73.0) months. Thirty-four patients were deemed evaluable for efficacy analysis, with 28 (82.4%) undergoing curative resection. This study was performed at 4 referral institutions in the US. Interventions: Patients received 3 cycles of capecitabine, 625 mg/m2, orally twice daily for 21 days; oxaliplatin, 130 mg/m2, intravenously and pembrolizumab, 200 mg, intravenously with optional epirubicin, 50 mg/m2, every 3 weeks before and after surgery with an additional cycle of pembrolizumab before surgery. Patients received 14 additional doses of maintenance pembrolizumab. Main Outcomes and Measures: The primary end point was pathologic complete response (pCR) rate. Secondary end points included overall response rate, disease-free survival (DFS), overall survival (OS), and safety. Results: A total of 34 patients (median [range] age, 65.5 [25-90] years; 23 [67.6%] male) were evaluable for efficacy. Of these patients, 28 (82.4%) underwent curative resection, 7 (20.6%; 95% CI, 10.1%-100%) achieved pCR, and 6 (17.6%) achieved a pathologic near-complete response. Of the 28 patients who underwent resection, 4 (14.3%) experienced disease recurrence. The median DFS and OS were not reached. The 2-year DFS was 67.8% (95% CI, 0.53%-0.87%) and the OS was 80.6% (95% CI, 0.68%-0.96%). Treatment-related grade 3 or higher adverse events for evaluable patients occurred in 20 patients (57.1%), and 12 (34.3%) experienced immune-related grade 3 or higher adverse events. Conclusion and Relevance: In this trial of unselected patients with resectable G/GEJ adenocarcinoma, capecitabine, oxaliplatin, and pembrolizumab resulted in a pCR rate of 20.6% and was well tolerated. This trial met its primary end point and supports the development of checkpoint inhibition in combination with perioperative chemotherapy in locally advanced G/GEJ adenocarcinoma. Trial Registration: ClinicalTrials.gov Identifier: NCT02918162.

Multiploid inheritance of HIV-1 during cell-to-cell infection.

During cell-to-cell transmission of human immunodeficiency virus type 1 (HIV-1), many viral particles can be simultaneously transferred from infected to uninfected CD4 T cells through structures called virological synapses (VS). Here we directly examine how cell-free and cell-to-cell infections differ from infections initiated with cell-free virus in the number of genetic copies that are transmitted from one generation to the next, i.e., the genetic inheritance. Following exposure to HIV-1-expressing cells, we show that target cells with high viral uptake are much more likely to become infected. Using T cells that coexpress distinct fluorescent HIV-1 variants, we show that multiple copies of HIV-1 can be cotransmitted across a single VS. In contrast to cell-free HIV-1 infection, which titrates with Poisson statistics, the titration of cell-associated HIV-1 to low rates of overall infection generates a constant fraction of the newly infected cells that are cofluorescent. Triple infection was also readily detected when cells expressing three fluorescent viruses were used as donor cells. A computational model and a statistical model are presented to estimate the degree to which cofluorescence underestimates coinfection frequency. Lastly, direct detection of HIV-1 proviruses using fluorescence in situ hybridization confirmed that significantly more HIV-1 DNA copies are found in primary T cells infected with cell-associated virus than in those infected with cell-free virus. Together, the data suggest that multiploid inheritance is common during cell-to-cell HIV-1 infection. From this study, we suggest that cell-to-cell infection may explain the high copy numbers of proviruses found in infected cells in vivo and may provide a mechanism through which HIV preserves sequence heterogeneity in viral quasispecies through genetic complementation.

Histopathology of Labyrinthitis Ossificans From an Intralabyrinthine Surgical Specimen.

OBJECTIVE: Histologic characterization of labyrinthitis ossificans (LO) has mostly been limited to postmortem samples. In this report, we describe the histology of LO from a surgical specimen obtained from a patient undergoing labyrinthectomy with simultaneous cochlear implantation. PATIENT: A 38-year-old woman initially presenting to the emergency room with acute vertigo, left-sided hearing loss, tinnitus, and aural fullness. INTERVENTIONS: Contrast-enhanced magnetic resonance imaging (MRI) and computerized tomography (CT) were performed for the patient before labyrinthectomy and cochlear implantation. Audiometric testing was performed before and after surgical intervention. Histologic analysis was performed on a specimen obtained from the left lateral semicircular canal (SCC) during surgery. MAIN OUTCOME MEASURES: Preoperative CT and MRI findings, preoperative and postoperative hearing thresholds via air conduction and bone conduction, hematoxylin and eosin (H&E) stain of the surgical sample, and CD45 immunostain of the surgical sample. RESULTS: Preoperative CT and MRI confirmed the diagnosis of isolated LO of the left lateral SCC. Audiometric testing revealed significant improvement in hearing after labyrinthectomy and cochlear implantation. H&E stain demonstrated fibrosis and ossification in the left lateral SCC, and CD45 immunostain was negative. CONCLUSIONS: Histopathology of LO based on a surgical sample is comparable to heterotopic ossification (HO) seen in other human tissue. Given the similarities between LO and HO, agents effective in preventing HO may have utility in preventing LO.

Invasive carcinoma versus pseudoinvasion: interobserver variability in the assessment of left-sided colorectal polypectomies.

OBJECTIVES: Misplaced epithelium in adenomas can occasionally be difficult to distinguish from invasive adenocarcinoma. We evaluated interobserver variability in the assessment of left-sided colon polypectomies for pseudoinvasion versus invasive adenocarcinoma and further investigated relevant histological findings. METHODS: 28 consecutive left-sided colon polyps with the keywords "pseudoinvasion", "epithelial misplacement", "herniation", "prolapse" or "invasive adenocarcinoma" were collected from 28 patients and reviewed by eight gastrointestinal pathologists. Participants assessed stromal hemosiderin, lamina propria/eosinophils surrounding glands, desmoplasia, high grade dysplasia/intramucosal adenocarcinoma and margin status and rendered a diagnosis of pseudoinvasion, invasive adenocarcinoma, or both. RESULTS: Agreement among pathologists was substantial for desmoplasia (κ=0.70), high grade dysplasia/intramucosal adenocarcinoma (κ=0.66), invasive adenocarcinoma (κ=0.63) and adenocarcinoma at the margin (κ=0.65). There was moderate agreement for hemosiderin in stroma (κ=0.53) and prolapse/pseudoinvasion (κ=0.50). Agreement was low for lamina propria/eosinophils around glands (κ=0.12). For invasive adenocarcinoma, seven or more pathologists agreed in 24 of 28 cases (86%), and there was perfect agreement in 19/28 cases (68%). For pseudoinvasion, seven or more pathologists agreed in 19 of 28 cases (68%), and there was perfect agreement in 16/28 cases (57%). CONCLUSION: Moderate to substantial, though imperfect, agreement was achieved in the distinction of pseudoinvasion from invasive carcinoma.

A 52-Year-Old Man With Chest Pain and Dyspnea.

CASE PRESENTATION: A 52-year-old man came to the cardiac surgery clinic for pulmonary thromboendarterectomy (PTE) evaluation. He had initially appeared at an outside hospital 1 year earlier, with chest pain and shortness of breath. He had no known chronic conditions. A CT pulmonary angiogram (CTPA) at that time showed a filling defect at the bifurcation of the main pulmonary artery. A transthoracic echocardiogram revealed mild mitral valve regurgitation, but otherwise the results were normal. As he was hemodynamically stable and not hypoxemic, he was treated solely by anticoagulation. Despite adhering to prescribed apixaban, he developed progressive dyspnea and reduced exercise tolerance over the subsequent year. A repeat CTPA performed 12 months after the initial presentation showed a persistent filling defect at the level of the pulmonary artery bifurcation, with a new extension now completely occluding the right main pulmonary artery. A pulmonary angiogram confirmed this complete occlusion, and right heart catheterization revealed precapillary pulmonary hypertension, with a mean pulmonary artery pressure of 50 mm Hg. His anticoagulation was transitioned to enoxaparin for presumed apixaban treatment failure, and an investigation for hypercoagulable conditions was initiated. His lupus anticoagulant test result was positive, but he did not meet the criteria for antiphospholipid syndrome because he was negative for anticardiolipin and ß2-glycoprotein antibodies. Assays for antithrombin III, protein C, prothrombin gene, and factor V Leiden mutations produced normal results.

Multiplexed single-cell analysis reveals prognostic and nonprognostic T cell types in human colorectal cancer.

Clinical outcomes in colorectal cancer (CRC) correlate with T cell infiltrates, but the specific contributions of heterogenous T cell types remain unclear. To investigate the diverse function of T cells in CRC, we profiled 37,931 T cells from tumors and adjacent normal colon of 16 patients with CRC with respect to transcriptome, TCR sequence, and cell surface markers. Our analysis identified phenotypically and functionally distinguishable effector T cell types. We employed single-cell gene signatures from these T cell subsets to query the TCGA database to assess their prognostic significance. We found 2 distinct cytotoxic T cell types. GZMK+KLRG1+ cytotoxic T cells were enriched in CRC patients with good outcomes. GNLY+CD103+ cytotoxic T cells with a dysfunctional phenotype were not associated with good outcomes, despite coexpression of CD39 and CD103, markers that denote tumor reactivity. We found 2 distinct Treg subtypes associated with opposite outcomes. While total Tregs were associated with good outcomes, CD38+ Tregs were associated with bad outcomes independently of stage and possessed a highly suppressive phenotype, suggesting that they inhibit antitumor immunity in CRC. These findings highlight the potential utility of these subpopulations in predicting outcomes and support the potential for novel therapies directed at CD38+ Tregs or CD8+CD103+ T cells.

Doxycycline-induced cholestatic liver injury.

Doxycycline-induced liver injury is a rare phenomenon, with an unclear clinical course and etiopathogenesis. The onset of injury may be acute-to-subacute, with a pattern ranging from hepatocellular or cholestatic to mixed, and it often lasts up to several weeks. We present a case of cholestatic liver injury secondary to doxycycline use in a middle-aged woman. In patients with a history of doxycycline exposure and subsequent hepatic injury, an adverse drug reaction due to doxycycline should remain on the differential, and immediate removal of the offending agent with close monitoring of the clinical condition should be pursued.

SATB2 in Neoplasms of Lung, Pancreatobiliary, and Gastrointestinal Origins.

OBJECTIVES: Special AT-rich binding protein 2 (SATB2) immunohistochemistry (IHC) has high sensitivity and specificity for colorectal adenocarcinoma (CRC), but data on its expression in specific subsets of pulmonary, gastric, small bowel, and pancreatobiliary adenocarcinomas (ADCAs) are relatively limited or discordant. We assessed SATB2 expression in a large cohort of ADCAs from these sites to determine its reliability in distinguishing CRC from them. METHODS: SATB2 IHC was performed on 335 neoplasms, including 40 lung ADCAs, 165 pancreatobiliary neoplasms (34 intraductal papillary mucinous neoplasms [IPMNs], 19 pancreatic ADCAs, 112 cholangiocarcinomas [CCs]), and 35 gastric, 13 small bowel, 36 ampullary (AMP), and 46 CRC ADCAs. The cases were evaluated for positivity (defined as ≥5% nuclear staining), and an H-score was calculated based on the percentage of SATB2+ cells and staining intensity. Analysis was performed to determine the optimal H-score threshold to separate CRC and non-CRC. RESULTS: SATB2 was positive in 3% of lung, 2% of CC, 17% of gastric, 38% of small bowel, and 6% of AMP ADCAs. All pancreatic ADCA/IPMNs were negative, and 87% CRCs were positive. CONCLUSIONS: SATB2 is not entirely specific for colorectal origin and can be expressed in a subset of gastrointestinal ADCAs. It is most useful in the differential of CRC vs lung and pancreatobiliary ADCAs.

Randomized Controlled Trial of the Gastrin/CCK2 Receptor Antagonist Netazepide in Patients with Barrett's Esophagus.

Hypergastrinemia has been associated with high-grade dysplasia and adenocarcinoma in patients with Barrett's esophagus, and experimental studies suggest proinflammatory and proneoplastic effects of gastrin on Barrett's esophagus. This is of potential concern, as patients with Barrett's esophagus are treated with medications that suppress gastric acid production, resulting in increased physiologic levels of gastrin. We aimed to determine whether treatment with the novel gastrin/CCK2 receptor antagonist netazepide reduces expression of markers associated with inflammation and neoplasia in Barrett's esophagus. This was a randomized, double-blind, placebo-controlled trial of netazepide in patients with Barrett's esophagus without dysplasia. Subjects were treated for 12 weeks, with endoscopic assessment at baseline and at end of treatment. The primary outcome was within-individual change in cellular proliferation as assessed by Ki67. Secondary analyses included changes in gene expression, assessed by RNA-sequencing, and safety and tolerability. A total of 20 subjects completed the study and were included in the analyses. There was no difference between arms in mean change in cellular proliferation (netazepide: +35.6 Ki67+ cells/mm2, SD 620.7; placebo: +307.8 Ki67+ cells/mm2, SD 640.3; P = 0.35). Netazepide treatment resulted in increased expression of genes related to gastric phenotype (TFF2, MUC5B) and certain cancer-associated markers (REG3A, PAX9, MUC1), and decreased expression of intestinal markers MUC2, FABP1, FABP2, and CDX1 No serious adverse events related to study drug occurred. The gastrin/CCK2 receptor antagonist netazepide did not reduce cellular proliferation in patients with nondysplastic Barrett's esophagus. Further research should focus on the biological effects of gastrin in Barrett's esophagus.Prevention Relevance: Treatment of patients with Barrett's esophagus with a gastrin/CCK2 receptor antagonist did not have obvious chemopreventive effects.

Update on risk stratification in the Papanicolaou Society of Cytopathology System for Reporting Pancreaticobiliary Cytology categories: 3-Year, prospective, single-institution experience.

BACKGROUND: Risk stratification is a critical element for the successful implementation of cytopathology reporting systems. To the authors' knowledge, there are limited prior studies regarding risk stratification for The Papanicolaou Society of Cytopathology System for Reporting Pancreaticobiliary Cytology (PSCPC). In the current study, the authors reported on a single-institution experience on 3-year prospective PSCPC regarding risk of malignancy (ROM) and the overall risk of malignancy (OROM). METHODS: A computerized search was performed from August 2014 to December 2017 for all pancreatic fine-needle aspiration (FNA) samples. Pathology from surgical resections and biopsies and relevant radiologic and clinical follow-up data were collected. The ROM and the OROM were calculated. The OROM was based on the total number of FNA samples in each category. RESULTS: A total of 1017 pancreatic FNA cases were identified, with surgical and/or clinical follow-up data available for 548 cases. The cytopathologic diagnoses included 242 nondiagnostic (category I), 162 benign (category II), 142 atypical (category III), 20 neoplastic-benign (category IV: benign), 133 neoplastic-other (category IV: other), 28 suspicious (category V), and 290 malignant (category VI) cases. A total of 364 malignancies were documented in 11 cases, 4 cases, 36 cases, 0 cases, 36 cases, 21 cases, and 255 cases, respectively, from categories I, II, III, IV: benign, IV:other, V, and VI. The ROM was 25%, 17.4%, 41.8%, 0%, 34.3% (95.2%), 95.5%, and 99.6%, respectively, and the OROM was 4.5%, 2.5%, 25.3%, 0%, 27.1% (83.3%), 75%, and 87.9%, respectively, for categories I, II, III, IV: benign, IV: other (with high-grade dysplasia), V, and VI. CONCLUSIONS: The true ROM for PSCPC is likely between the ROM and OROM for the benign and indeterminate categories. In the neoplastic-other category (category IV: other), identifying high-grade dysplasia is important for its association with malignancy and a higher ROM.