Diagnostic interobserver agreement for thyroid fine-needle aspirates: Effects of reviewer experience and molecular diagnostics.

OBJECTIVES: Few cytologically indeterminate thyroid fine-needle aspirations (FNAs) harbor BRAF V600E. Here, we assess interobserver agreement for The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) category III (atypia of undetermined significance [AUS]) FNAs harboring BRAF V600E and contrast their features with those harboring non-BRAF V600E alterations, with attention to cytopathology experience. METHODS: Seven reviewers evaluated 5 AUS thyroid FNAs harboring BRAF V600E. To blind reviewers, cases were intermixed with 19 FNAs falling within other TBSRTC categories and in which genetic alterations other than BRAF V600E had been identified (24 FNAs total). Interobserver agreement against both "index" and most popular ("mode") diagnoses was calculated. Four additional BRAF V600E cases were independently reviewed. RESULTS: Reviewers included 3 trainees and 3 American Board of Pathology (board)-certified cytopathologists. Board-certified cytopathologists, whose experience ranged from 2 to more than 15 subspecialty practice years, had known AUS rates. BRAF V600E was identified in 5 of 260 (2%) AUS FNAs. Interobserver agreement was higher among cytopathologists with more experience. Mode diagnosis differed from index diagnosis in 6 of 11 cases harboring RAS-like alterations; mode diagnosis was AUS in 4 of 5 BRAF V600E FNAs. CONCLUSIONS: Atypia of undetermined significance of thyroid FNAs harboring BRAF V600E is uncommon yet relatively reproducible, particularly among pathologists with experience. It is advisable to sequence BRAF across V600 in such cases.

Interval Appendectomy Specimens.

CONTEXT.­: Patients with perforated appendicitis are often managed with antibiotic therapy followed by a delayed appendectomy. Histologic features of such specimens have been incompletely described, especially in the recent literature. OBJECTIVE.­: To describe the histomorphology of interval appendicitis with a focus on features that could mimic important conditions, such as infections, Crohn disease, and mucinous neoplasms. DESIGN.­: Histologic evaluation of 100 interval appendectomy specimens with clinical and radiologic correlation. RESULTS.­: A total of 54 of the 100 patients (54%) had radiologic evidence of appendiceal perforation, and 97% were treated with intravenous and/or oral antibiotic therapy prior to appendectomy. Percutaneous drains were placed in 34 cases (34%). Common histologic findings included mural eosinophilic infiltration (54%), periappendiceal fibrosis (54%), and xanthogranulomatous inflammation (31%). Periappendiceal fibrosis was frequent among patients with radiologic evidence of perforation. Nine cases (9%) featured pulse granulomata associated with fecal material. Epithelioid granulomata were detected in 6% of cases and were confined to mucosal lymphoid follicles in all cases. Only 4 of these were accompanied by mural lymphoid aggregates that raised the possibility of Crohn disease. Changes mimicking mucinous neoplasms were more common: 14% of cases (14 of 100) displayed goblet cell hyperplasia, 15% (15 of 100) contained diverticula, and 16% (16 of 100) showed mural or periappendiceal mucin pools. CONCLUSIONS.­: Although interval appendectomy specimens occasionally contain inflammatory infiltrates that mimic infections and/or Crohn disease, changes that can be confused with mucinous neoplasms are more frequently encountered.

Combining molecular testing and the Bethesda category III:VI ratio for thyroid fine-needle aspirates: A quality-assurance metric for evaluating diagnostic performance in a cytopathology laboratory.

BACKGROUND: Molecular testing (MT) of thyroid fine-needle aspiration (FNA)-derived genetic material is commonly used to assess malignancy risk for indeterminate cases. The Bethesda System for Reporting Thyroid Cytopathology (TBS) provides limited guidance for the appropriate use of category III (atypia of undetermined significance [AUS]). The authors combined MT with cytomorphology to monitor AUS diagnoses in a cytopathology laboratory. METHODS: Neoplasia-associated genetic alterations (NGAs) were determined by MT of preoperative FNA biopsies or resected malignancies and were categorized as BRAF V600E mutations, RAS-like mutations (HRAS, NRAS, or KRAS mutations or non-V600E BRAF mutations), or other mutations. RESULTS: Among 7382 thyroid FNA biopsies, the AUS rate was 9.3% overall and ranged from 4.3% to 24.2% among 6 cytopathologists (CPs) who evaluated >150 cases. The ratio of specimens falling into TBS category III to specimens falling into category VI (malignant) (the III:VI ratio) was 2.4 overall (range, 1.1-8.1), and the ratio of specimens falling into TBS categories III and IV (follicular neoplasm or suspicious for follicular neoplasm) combined (III+IV) to specimens falling into category VI (the [III+IV]:VI ratio) was 2.9 overall (range, 1.4-9.5). MT was performed on 588 cases from 560 patients (79% women) with a median age of 56 years (range, 8-89 years). BRAF V600E mutation was the most common (76% of cases) in TBS category VI and was rare (3%) in category III. RAS-like mutations were most common in TBS categories III (13%), IV (25%), and V (suspicious for malignancy) (17.5%). The NGA rate in AUS cases fell between 5% and 20% for 5 of 6 CPs and did not correlate with the III:VI ratio or the (III+IV):VI ratio. CONCLUSIONS: Lack of correlation between the NGA rate and easily calculable diagnostic ratios enables the calibration of diagnostic thresholds, even for CPs who have normal metrics. Specifically, calculation of the NGA rate and the III:VI ratio may allow individual CPs to determine whether they are overcalling or undercalling cases that other CPs might otherwise recategorize.

Histologic Features of Tacrolimus-induced Colonic Injury.

Tacrolimus is a common immunosuppressant used in solid organ transplant recipients. Although most patients develop diarrheal symptoms, data regarding patterns of injury in patients taking tacrolimus are limited. We performed this study to characterize tacrolimus-related features of colonic injury. We retrospectively identified colonic samples from 20 patients receiving tacrolimus monotherapy. Records were reviewed for symptoms, endoscopic findings, other medications, and infections. None of the patients had gastrointestinal infections or used other drugs known to cause colonic injury; none had received mycophenolate within 6 months of presentation. Cases were evaluated for the nature and distribution of inflammation and crypt abnormalities, including distortion, destruction, and apoptosis. Eighteen (90%) patients were solid organ transplant recipients. Seventeen (85%) had gastrointestinal symptoms, particularly diarrhea (75%). More than 50% had endoscopic colitis and 15% had ulcers and/or erosions. Most (90%) cases showed regenerative epithelial changes; apoptotic crypt cells were present in 55% and numerous in 10% of cases. Neutrophilic cryptitis was present in 60% of cases; 35% showed crypt destruction. Plasma cell-rich lamina propria inflammation and crypt distortion were observed in 40% and 25% of cases, respectively. There was no correlation between therapy duration and features of chronic injury. We conclude that tacrolimus can cause symptomatic colitis. Histologic abnormalities are often mild, featuring regenerative crypts and scattered apoptotic debris. However, 40% of symptomatic patients have chronic colitis, most likely reflecting drug-induced immune dysregulation. Pathologists should be aware of these associations because colitis often resolves with decreasing drug dosage rather than treatment directed toward inflammatory bowel disease.

Crohn Disease Infrequently Affects the Appendix and Rarely Causes Granulomatous Appendicitis.

Data from previous studies suggest Crohn disease of the appendix accounts for ∼25% of granulomatous appendicitis cases. However, we have found that granulomatous inflammation in appendectomy specimens rarely heralds Crohn disease. We suspect that appendiceal involvement by Crohn disease is uncommon, even when patients have severe ileocolonic inflammation. We performed this study to determine the prevalence and nature of appendiceal inflammation among patients with Crohn disease. We reviewed 100 ileocolic specimens with strictures and fistulizing Crohn disease for the nature and distribution of inflammatory changes in the appendix and compared them with 100 appendices on colectomy specimens from age-matched and sex-matched patients with ulcerative colitis. We also evaluated 27 additional cases of granulomatous appendicitis in appendectomy specimens to determine the frequency with which this finding represented Crohn disease. The appendix was usually normal (26%) or showed fibrous obliteration (50%) in ileocolic resection specimens from patients with Crohn disease. Mucosal inflammation was much less common in appendices from patients with Crohn disease than ulcerative colitis (6% vs. 28%, P<0.0001); only 4 cases contained epithelioid granulomata, 3 showed mural fibrosis and lymphoid aggregates, and 10 displayed only periappendiceal inflammation. None of the patients with granulomatous appendicitis in appendectomy specimens had, or developed, evidence of Crohn disease. We conclude that Crohn disease infrequently affects the appendix. Interval appendectomy and infection are more important considerations when appendectomy specimens feature granulomatous inflammation and/or mural lymphoid aggregates, especially if there is no history of idiopathic inflammatory bowel disease.

Autopsy Findings in 32 Patients with COVID-19: A Single-Institution Experience.

BACKGROUND: A novel coronavirus, SARS-CoV-2, was identified in Wuhan, China in late 2019. This virus rapidly spread around the world causing disease ranging from minimal symptoms to severe pneumonia, which was termed coronavirus disease (i.e., COVID). Postmortem examination is a valuable tool for studying the pathobiology of this new infection. METHODS: We report the clinicopathologic findings from 32 autopsy studies conducted on patients who died of COVID-19 including routine gross and microscopic examination with applicable special and immunohistochemical staining techniques. RESULTS: SARS-CoV-2 infection was confirmed by nasopharyngeal RT-PCR in 31 cases (97%) and by immunohistochemical staining for SARS-CoV-2 spike-protein in the lung in the remaining 1 case (3%). The ethnically diverse cohort consisted of 22 males and 10 females with a mean age of 68 years (range: 30-100). Patients most commonly presented with cough (17 [55%]), shortness of breath (26 [81%]), and a low-grade fever (17 [55%]). Thirty-one (97%) of the patients had at least 1 comorbidity (mean = 4). Twenty-eight patients (88%) had widespread thromboembolic disease, as well as diffuse alveolar damage (30 [94%]), diabetic nephropathy (17 [57%]) and acute tubular injury. Patterns of liver injury were heterogeneous, featuring 10 (36%) with frequent large basophilic structures in sinusoidal endothelium, and increased immunoblast-like cells in lymph nodes. CONCLUSION: This series of autopsies from patients with COVID-19 confirms the observation that the majority of severely affected patients have significant pulmonary pathology. However, many patients also have widespread microscopic thromboses, as well as characteristic findings in the liver and lymph nodes.

Clinicopathologic Features of Varicella Zoster Virus Infection of the Upper Gastrointestinal Tract.

Reactivation of latent varicella zoster virus (VZV) may be limited to a dermatome or involve multiple organs, including the gastrointestinal tract. Although gastrointestinal manifestations of disseminated zoster have been likened to those of herpes simplex virus (HSV), histologic features of VZV-related injury to the tubular gut are not well-documented. We performed this study to describe the clinicopathologic features of VZV-related gastrointestinal injury. We identified 6 such patients with VZV infection. All involved the upper gastrointestinal tract, affecting the esophagus (n=3), stomach (n=2), or both (n=1). All patients were immunocompromised adults with hematologic malignancies (n=5) or a heart transplant (n=1); 3 with hematologic malignancies had received stem cell transplants. Five patients had cutaneous and gastrointestinal zoster; 1 had gastrointestinal disease alone. When compared with 14 HSV-related esophagitis controls, there were several notable differences. VZV caused hemorrhagic ulcers with nodularity or erythema, whereas HSV produced round, shallow ulcers on a background of nearly normal mucosa (P=0.01). VZV-related ulcers featured fibrin-rich, pauci-inflammatory exudates compared with the macrophage-rich exudates of HSV (P=0.003). The cytopathic changes of VZV were present at all levels of the squamous epithelium, especially in a peripapillary distribution. In contrast, HSV inclusions were located in the superficial layers (P=0.003) and detached keratinocytes. Unlike HSV, VZV involved the stomach, producing hemorrhage accompanied by striking apoptosis in the deep glands. We conclude that VZV produces unique patterns of gastrointestinal injury that facilitate its diagnosis. Recognition of gastrointestinal VZV infection is important because it heralds potentially life-threatening disseminated disease.

COVID-19 pulmonary pathology: a multi-institutional autopsy cohort from Italy and New York City.

SARS-CoV-2, the etiologic agent of COVID-19, is a global pandemic with substantial mortality dominated by acute respiratory distress syndrome. We systematically evaluated lungs of 68 autopsies from 3 institutions in heavily hit areas (2 USA, 1 Italy). Detailed evaluation of several compartments (airways, alveolar walls, airspaces, and vasculature) was performed to determine the range of histologic features. The cohort consisted of 47 males and 21 females with a median age of 73 years (range 30-96). Co-morbidities were present in most patients with 60% reporting at least three conditions. Tracheobronchitis was frequently present, independent from intubation or superimposed pneumonia. Diffuse alveolar damage (DAD) was seen in 87% of cases. Later phases of DAD were less frequent and correlated with longer duration of disease. Large vessel thrombi were seen in 42% of cases but platelet (CD61 positive) and/or fibrin microthrombi were present at least focally in 84%. Ultrastructurally, small vessels showed basal membrane reduplication and significant endothelial swelling with cytoplasmic vacuolization. In a subset of cases, virus was detected using different tools (immunohistochemistry for SARS-CoV-2 viral spike protein, RNA in situ hybridization, lung viral culture, and electron microscopy). Virus was seen in airway epithelium and type 2 pneumocytes. IHC or in situ detection, as well as viable form (lung culture positive) was associated with the presence of hyaline membranes, usually within 2 weeks but up to 4 weeks after initial diagnosis. COVID-19 pneumonia is a heterogeneous disease (tracheobronchitis, DAD, and vascular injury), but with consistent features in three centers. The pulmonary vasculature, with capillary microthrombi and inflammation, as well as macrothrombi, is commonly involved. Viral infection in areas of ongoing active injury contributes to persistent and temporally heterogeneous lung damage.

Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome.

COVID-19 affects millions of patients worldwide, with clinical presentation ranging from isolated thrombosis to acute respiratory distress syndrome (ARDS) requiring ventilator support. Neutrophil extracellular traps (NETs) originate from decondensed chromatin released to immobilize pathogens, and they can trigger immunothrombosis. We studied the connection between NETs and COVID-19 severity and progression. We conducted a prospective cohort study of COVID-19 patients (n = 33) and age- and sex-matched controls (n = 17). We measured plasma myeloperoxidase (MPO)-DNA complexes (NETs), platelet factor 4, RANTES, and selected cytokines. Three COVID-19 lung autopsies were examined for NETs and platelet involvement. We assessed NET formation ex vivo in COVID-19 neutrophils and in healthy neutrophils incubated with COVID-19 plasma. We also tested the ability of neonatal NET-inhibitory factor (nNIF) to block NET formation induced by COVID-19 plasma. Plasma MPO-DNA complexes increased in COVID-19, with intubation (P < .0001) and death (P < .0005) as outcome. Illness severity correlated directly with plasma MPO-DNA complexes (P = .0360), whereas Pao2/fraction of inspired oxygen correlated inversely (P = .0340). Soluble and cellular factors triggering NETs were significantly increased in COVID-19, and pulmonary autopsies confirmed NET-containing microthrombi with neutrophil-platelet infiltration. Finally, COVID-19 neutrophils ex vivo displayed excessive NETs at baseline, and COVID-19 plasma triggered NET formation, which was blocked by nNIF. Thus, NETs triggering immunothrombosis may, in part, explain the prothrombotic clinical presentations in COVID-19, and NETs may represent targets for therapeutic intervention.

Hidden Carcinoma: Pitfalls in the Diagnosis of Lymphoepithelioma-Like Cholangiocarcinoma.

Lymphoepithelioma-like intrahepatic cholangiocarcinoma is a rare variant of cholangiocarcinoma that is associated with the Epstein-Barr virus. The intimate relationship between the malignant epithelial cells and the numerous lymphoid cells can make the diagnosis challenging on limited tissue samples. We present 2 cases in which the presence of a dense hematolymphoid infiltrate served to mask the diagnosis of carcinoma on initial frozen section and biopsy review, respectively. We bring awareness to this potential diagnostic pitfall and offer morphologic and immunohistochemical clues that may aid in recognition of this unusual and sometimes perplexing carcinoma.

Sarcoid-Like Granulomatosis in a Patient With Breast Cancer Mimicking Refractory Metastatic Disease.

Sarcoid-like granulomatosis is a known but rare adverse reaction to immune checkpoint inhibitors and chemotherapy in the treatment of advanced solid tumors. We present a case of a 29-year-old female with a pathologically confirmed poorly differentiated invasive ductal carcinoma of the breast with presumed metastases to the lungs, hilar lymph nodes, liver, and spleen. Despite appropriate chemotherapy, the patient developed pulmonary lesions that were interpreted on imaging studies as progression of malignancy. Autopsy revealed disseminated sarcoid-like granulomatosis with multiple noncaseating granulomata with associated fibrosis in the lungs, liver, and spleen. No residual invasive carcinoma or metastatic disease was identified. This case illustrates the difficulty in differentiating this nonneoplastic process from progressive disease in the clinical setting.