INTRODUCTION:Despite imaging advancements, proper management for cardiac amyloidosis hinges on accurate diagnosis and subtyping of amyloid in endomyocardial biopsies (EMBx). Nonetheless, the approach toward identifying amyloid in EMBx varies among pathologists. Various staining techniques can be used, each with its own advantages and limitations. There is a lack of evidence-based data to compare different methodologies to detect amyloid in EMBx. In this study, we compared the efficacy of H&E stains only, Congo red (bright field, polarized, and fluorescence), trichrome, and Thioflavin S for the diagnosis of amyloid in EMBx. We also validated a digital imaging workflow for Congo red (brightfield, polarized, and fluorescence microscopy) and Thioflavin S in EMBx. METHODS:We retrospectively identified 40 EMBx (21 positive and 19 negative) that were evaluated for amyloid deposition. Each biopsy was stained with H&E, Congo red, trichrome, and Thioflavin S. The slides were separated into three independently randomized and blinded cohorts: HE + Congo red, HE + trichrome, and HE + thioflavin S. Cases were evaluated for amyloid deposition by three cardiothoracic pathologists (CTP) and two pathologists-in-training (PIT) based on (1) H&E only (2) Congo red brightfield (3) Congo red polarized, (4) Congo red fluorescence, (5) trichrome, and (6) Thioflavin S. After a 3-month washout period, the Congo red and Thioflavin S slides were scanned using an Olympus VS200 slide scanner to capture brightfield, polarized, and fluorescent images, which were then reviewed by all reviewers. This validation was exempt from IRB review. RESULTS:Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for each stain when reviewed by CTP alone as well as CTP+PIT. When glass slides were reviewed by CTP + PIT, sensitivity ranged from 86.7%-99.1%, specificity 93.6%-99.0%, PPV 94.4%-99.1%, and NPV 86.8%-99.0%. Statistical analysis revealed no significant differences in sensitivity, specificity, PPV, or NPV between Congo red polarized and any other studied stain. When digital images were reviewed by CTP + PIT, sensitivity ranged from 88.6%-97.1%, specificity 91.6%-100%, PPV 92.2%-100%, and NPV 88.6%-96.9%. Statistical analysis revealed no significant differences between stains interpreted by digital imaging and their corresponding glass slides. CONCLUSION:Congo red staining with polarized microscopy is often considered the gold standard for detecting amyloid deposits. We demonstrated that trichrome and Thioflavin S stains show comparable results and can be considered as appropriate methods for diagnosis. In addition, reviewing Congo red brightfield, polarized, and fluorescence as well as Thioflavin S with digital imaging shows a comparable diagnostic yield to glass slides for identification of cardiac amyloid deposition. These findings suggest that a digital workflow can be implemented for diagnosing cardiac amyloid deposition in routine practice.

The consequence of lymphatic disruption during transplantation of solid organs remains unknown. Long-term survival after organ transplantation is limited by chronic rejection, a poorly understood process involving fibrotic remodeling and functional decline of the graft. Here, we found that transplanted human lungs and hearts with chronic rejection exhibited fibrosis distributed along dysmorphic lymphatics in areas densely concentrated with hyaluronan, an interstitial glycosaminoglycan that depends on lymphatic drainage for clearance. We illustrated similar findings in transplanted mouse lungs and hearts, which were accompanied by lymphographic findings of graft lymphedema. Using unsupervised clustering, we found a subset of stromal cells present in fibrotic syngeneic mouse lung grafts and human lung and heart grafts with chronic rejection that coexpressed hyaluronan synthase 1 and interleukin-1 receptor 1. Shortly after reperfusion of syngeneic mouse lung grafts, we identified neutrophilic expression of interleukin-1β (Il1b) as a driver of hyaluronan synthase 1 up-regulation. We found interleukin-1-mediated hyaluronan accumulation as a mechanism driving fibrosis that occurred independent of alloimmunity in the setting of lymphatic disruption after transplantation. Development of fibrotic remodeling in transplanted mouse lungs was inhibited by preventing hyaluronan synthesis through the administration of 4-methylumbilliferone, accelerating lymphangiogenesis with pharmacologic activation of VEGF (vascular endothelial growth factor) receptor-3, or inhibiting interleukin-1 receptor 1 signaling in the graft. These therapeutic interventions lay the foundation for future clinical strategies to prevent chronic rejection.

Introduction The Society for Cardiovascular Pathology (SCVP) recently released a consensus statement on diagnosing and reporting temporal artery biopsies (TAB). The document addresses diagnostic challenges such as adventitial inflammation, absence of giant cells, and non-arteritis changes. It recommends a three-tiered diagnostic scheme and emphasizes recognizing other types of vasculitis although giant cell arteritis (GCA) is the most common type. In this study, we retrospectively reviewed archival TABs to evaluate the utility of this new statement. Materials and methods We identified all TABs from our files from 1/2022-12/2023. Slides were blindly reviewed without knowing the original diagnoses. Cases were categorized as active arteritis, no arteritis, or healed arterial injury per the consensus document. Active arteritis cases were further classified as GCA or non-GCA, and the presence of giant cells was noted. Results were compared with the original diagnoses to assess the diagnostic and reporting utility of the SCVP schema. Results We included 105 TABs from 102 patients (66 female, 36 male; ages 45-93 years; mean age 71 ± 10 years). Using the consensus guidelines, 12 cases were diagnosed as active arteritis. Notably, 8 of 12 cases did not display giant cells in the media, and 1 case showed an exuberant number of eosinophils, suggesting that non-GCA arteritis should be considered. These 12 cases plus another 2 were originally diagnosed as GCA; the latter 2 cases were reclassified as no arteritis, with a comment noting adventitial-only inflammation. Two additional cases were classified as no arteritis, with original descriptive diagnosis of chronic inflammation in only adventitia. The remaining 89 cases were diagnosed as no arteritis, consistent with the original diagnosis. We did not find any case of healed arterial injury. The most common non-arteritis findings were neointimal hyperplasia and internal elastic lamina calcifications, consistent with age-related changes. Conclusion The new SCVP TAB consensus schema can be easily implemented in routine clinical practice. It is also helpful in formulating diagnoses for challenging cases, such as those with adventitial-only inflammation or non-GCA arteritis. Additionally, it provides a unified and consistent reporting scheme.

INTRODUCTION:Heart transplantations are lifesaving for patients with end-stage heart failure. It is pertinent for the multidisciplinary care team to understand how heart transplant patients succumbed to death and the complications that occurred. In this study, we performed a comprehensive retrospective review of all the autopsies performed in our institute for heart transplant patients and report the trend of demographic data, cause of death, and autopsy findings. MATERIALS AND METHODS:Reports, photos, and slides of autopsies performed at our institute from 1990 to 2023 for heart transplant patients were reviewed. Pertinent demographic data (age, gender, pretransplant diagnosis), clinical data (clinical history of rejection, complication, time interval from transplant to death, clinical cause of death) and pathological findings (allograft pathology, infectious etiology, other findings related to cause of death) were reviewed, documented, and analyzed. RESULTS:We identified 88 cases, consisting of 53 male and 35 female patients. The median age at transplant was 26 years, while 28.5 years was the median age at death. The median interval from transplant to death was 10 months. The cases were classified in three categories based on length of survival post-transplant: Superacute (<1 month, 21%), Early (1 month-12 months, 30%), and Late (> 12 months, 49%). Slides were unavailable for review in 15 cases, which were excluded from cause of death (COD) evaluation. We categorized 41.1% of cases as allograft-related COD and 58.9% as non-allograft-related COD. Six of the CODs were not perceived premortem. These unexpected CODs included moderate/severe acute cellular rejection in a patient with a recently negative biopsy, dehiscent suture caused by a fungal abscess, an aorto-bronchial fistula, CMV myocarditis, acute abdominal bleeding, and ruptured atherosclerotic plaques with acute myocardial infarction. CONCLUSION:We systematically reviewed 33 years of heart transplant autopsies. We found that 41.1% of deaths were allograft related, with infection being the most frequent COD. While the rate of unexpected findings was low, the findings demonstrate the continued utility of autopsy in patient evaluation.

INTRODUCTION:Traditional Pap staining procedure involves immediate ethanol-fixation of prepared smears. When our department absorbed cytopathology services of an affiliate hospital using nonspecialized assistants, we had problems with poorly fixed slides. We evaluated an alternative approach of rehydration of air-dried slides in comparison to both immediate and delayed alcohol fixation for Pap staining. MATERIALS AND METHODS:Fine needle aspirations were performed on deidentified unfixed autopsy tissue. Sampled tissues included lung, liver, thyroid, breast, and a mediastinal mass. Paired aspirate smears were immediately fixed, air-dried and rehydrated before staining, or delayed fixation after a 5-minute air dry (DF). Additional smears were Diff-Quik stained for comparison. Slides were Pap-stained at 0 days, 1 day, 3 days, or 5 days postaspiration and numerically scored for cytomorphologic feature quality. Data were compared with one-way analysis of variance analysis. RESULTS:We evaluated 237 pairs of smears. The immediately fixed and air-dried groups displayed higher quality for nuclear borders, nuclear detail, distinct cell borders, and cytoplasmic staining at every time point (all P values <0.05) compared to the DF group. Rehydration did not reverse the air-dry artifact from delayed fixation in the DF group. CONCLUSIONS:Air-drying with delayed rehydration/alcohol fixation maintains diagnostic quality in settings for which traditional preparative techniques are not optimal, technical assistance is limited, and transport is problematic. Our series demonstrates that air-dried slides maintained high quality when processed up to 5 days postaspiration. We demonstrate a technique that improves pathology outreach by providing better quality diagnostic material with minimal additional personnel costs.

Context.—:Distinguishing benign from malignant processes in small lung biopsy specimens is challenging, particularly in the presence of architectural distortion and crush artifacts. Mimics include reactive type II pneumocytes in peribronchiolar metaplasia or organizing pneumonia and benign mucinous elements, including goblet cells and submucosal glands, which can mimic adenocarcinoma with mucinous features. Objective.—:To understand whether caudal-type homeobox 2 (CDX2) and cytokeratin 20 (CK20) immunoreactivity is specific to malignant epithelial processes and evaluate their expression in a range of benign, premalignant, and malignant pulmonary lesions. Design.—:We assessed CDX2 and CK20 immunohistochemistry in 48 cases of interstitial lung disease with reactive type II pneumocytes, 13 foci of atypical adenomatous hyperplasia, 11 bronchiolar adenomas, 4 adenocarcinomas in situ, 25 minimally invasive adenocarcinomas, 39 pulmonary adenocarcinomas with mucinous features, 6 mucoepidermoid carcinomas, and 1 squamous papilloma with goblet cell hyperplasia. Results.—:No expression of CDX2 or CK20 was observed in reactive processes, goblet cells, bronchial glands, atypical adenomatous hyperplasia, or bronchiolar adenomas. In adenocarcinomas with mucinous features (n = 39), 9 (23%) were CK20-reactive and 8 (20%) were CDX2-reactive. A minority of nonmucinous and mucinous minimally invasive adenocarcinomas (n = 25) showed immunoreactivity (3 [12%] for each marker), while all in situ adenocarcinomas were negative. Conclusions.—:Our findings demonstrate that CDX2 and CK20 are not expressed in benign or reactive type II pneumocytes and are specific for malignancy, particularly adenocarcinomas with mucinous features. Immunoreactivity with either marker in small biopsy specimens can support a diagnosis of malignancy, especially in morphologically equivocal or limited samples. Absence of staining does not exclude malignancy, but immunoreactivity with CDX2 and/or CK20 provides strong supportive evidence for a malignant process.

Deep learning-assisted digital pathology has demonstrated the potential to profoundly impact clinical practice, even surpassing human pathologists in performance. However, as deep neural network (DNN) architectures grow in size and complexity, their explainability decreases, posing challenges in interpreting pathology features for broader clinical insights into physiological diseases. To better assess the interpretability of digital microscopic images and guide future microscopic system design, we developed a novel method to study the predictive feature length-scale that underpins a DNN's predictive power. We applied this method to analyze a DNN's capability in predicting brain metastasis from early-stage non-small-cell lung cancer biopsy slides. This study quantifies DNN's attention for brain metastasis prediction, targeting features at both the cellular scale and tissue scale in H&E-stained histological whole slide images. At the cellular scale, the predictive power of DNNs progressively increases with higher resolution and significantly decreases when the resolvable feature length exceeds 5 microns. Additionally, DNN uses more macro-scale features associated with tissue architecture and is optimized when assessing visual fields greater than 41 microns. Our study computes the length-scale requirements for optimal DNN learning on digital whole-slide microscopic images, holding the promise to guide future optical microscope designs in pathology applications and facilitating downstream deep learning analysis.

Brain metastases can occur in nearly half of patients with early and locally advanced (stage I-III) non-small cell lung cancer (NSCLC). There are no reliable histopathologic or molecular means to identify those who are likely to develop brain metastases. We sought to determine if deep learning (DL) could be applied to routine H&E-stained primary tumor tissue sections from stage I-III NSCLC patients to predict the development of brain metastasis. Diagnostic slides from 158 patients with stage I-III NSCLC followed for at least 5 years for the development of brain metastases (Met+, 65 patients) versus no progression (Met-, 93 patients) were subjected to whole-slide imaging. Three separate iterations were performed by first selecting 118 cases (45 Met+, 73 Met-) to train and validate the DL algorithm, while 40 separate cases (20 Met+, 20 Met-) were used as the test set. The DL algorithm results were compared to a blinded review by four expert pathologists. The DL-based algorithm was able to distinguish the eventual development of brain metastases with an accuracy of 87% (p < 0.0001) compared with an average of 57.3% by the four pathologists and appears to be particularly useful in predicting brain metastases in stage I patients. The DL algorithm appears to focus on a complex set of histologic features. DL-based algorithms using routine H&E-stained slides may identify patients who are likely to develop brain metastases from those who will remain disease free over extended (>5 year) follow-up and may thus be spared systemic therapy. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

BACKGROUND:Diagnosis of mucinous carcinomas in the lung on transbronchial biopsy or fine-needle aspiration (FNA) samples can be difficult for the pathologist, because primary and metastatic tumors can have similar morphological, immunohistochemical, and molecular characteristics. Correct diagnosis is key to determine appropriate therapy and to distinguish primary from metastatic disease. This distinction often falls to the pathologist in patients with a history of mucinous adenocarcinoma of the colon. Despite its drawbacks, immunohistochemistry is often employed to help assign a primary site for mucinous adenocarcinomas in the lung. However, the published data in this regard is limited to studies that use only a handful of markers. METHODS:The authors examined the staining characteristics and heterogeneity of CK7, TTF-1, NapsinA, CK20, CDX2, and SATB2 in resection specimens of pulmonary adenocarcinomas with mucinous features and metastatic colorectal adenocarcinoma. RESULTS:Based on the heterogeneity, sensitivity, and specificity in this cohort, the authors developed a decision tree based on TTF-1, SATB2, CDX2, and CK7 to categorize tumors as primary or metastatic lesions. Validation of the decision tree in FNA specimens from the lungs and lung-draining lymph nodes showed 84% concurrence in cases from the lung and 100% concurrence in cases from the lymph node. In cases where the algorithm assigned a primary site, it was 95% accurate compared to the multidisciplinary diagnosis. CONCLUSIONS:This method holds promise in distinguishing primary versus metastatic lesions in resection, biopsy, and FNA samples from the lungs.

Human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) is a unique form of carcinoma that largely arises from the tonsillar tissue in the oropharynx. These tumors often present with cervical lymphadenopathy resulting in a fine needle aspiration (FNA) biopsy. Use of the cytology specimen to determine the HPV-status has significant prognostic and treatment implications as HPV-related tumors have a more favorable prognosis and response to nonsurgical therapies. While several different ancillary testing methods are available that have proven effective for determining HPV status in FNA specimens from HNSCCs, there is currently no consensus regarding HPV testing in this setting. Diagn. Cytopathol. 2017;45:221-229. © 2016 Wiley Periodicals, Inc.

BACKGROUNDHuman papillomavirus (HPV)‐related oropharyngeal squamous cell carcinoma (SCC) is a unique form of carcinoma that is important to identify for prognosis and treatment. Immunohistochemistry (IHC) for p16 (also known as cyclin‐dependent kinase inhibitor 2A, multiple tumor suppressor 1) is used as a surrogate marker for transcriptionally active, high‐risk HPV. The primary objective of this study was to correlate p16 IHC of cell blocks from fine‐needle aspirations (FNAs) with surgical pathology specimens of HPV‐related oropharyngeal SCC.METHODSIn total, 48 patients who had a diagnosis of oropharyngeal or nonoropharyngeal SCC and also had an FNA that demonstrated metastatic SCC with available cell block material were identified. IHC for p16 was evaluated on both FNA cell blocks and surgical pathology specimens. In situ hybridization for high‐risk HPV messenger RNA was performed on 31 of the FNA cell blocks.RESULTSAlthough partial p16 staining was observed in the majority of cell blocks, there was concordance in 47 of 48 FNAs (98%) with surgical pathology specimens when strong positive p16 staining of at least 15% of tumor cells in FNA cell block material was present. In addition, high‐risk HPV RNA in situ hybridization demonstrated a high correlation with p16 staining in surgical pathology specimens (96%) and FNAs (93%).CONCLUSIONSThere was excellent correlation between p16 IHC of FNA cell blocks and surgical pathology specimens using a cutoff of at least 15% positive staining in cell blocks. The recommended threshold (70% positive staining) for surgical pathology specimens may yield a high rate of false‐negative results if applied to FNA cell blocks. Cancer (Cancer Cytopathol) 2015;123:723–731. © 2015 American Cancer Society.

INTRODUCTION:Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (SCC) is a biologically unique form of carcinoma that is important to identify for prognosis and treatment. The objective of this study was to evaluate the performance of the Aptima HPV assay using Diff-Quick (DQ) stained smears from fine-needle aspiration (FNA) of HPV-related oropharyngeal SCC.MATERIALS AND METHODS:Patients with a diagnosis of head and neck SCC who also had FNA sample demonstrating metastatic disease were identified. Using a mounting media-based cell transfer technique, approximately 200 tumor cells were selected and harvested from DQ-stained aspirate smeared slides. The selected cells were tested for high risk HPV using the Aptima HPV assay, an in vitro nucleic acid amplification test for the qualitative detection of E6/E7 viral messenger RNA from high-risk types of HPV. These results were compared with the p16 immunohistochemical staining of the corresponding surgical pathology specimens.RESULTS:Twenty-eight of 32 (87.5%) FNAs of p16-positive oropharyngeal SCC were positive for high-risk HPV by the Aptima assay and 18 of 18 (100%) FNAs of p16-negative SCC were negative for high-risk HPV by the Aptima assay.CONCLUSIONS:DQ-stained FNA smears can be used by the Aptima HPV assay to accurately detect high-risk HPVs in oropharyngeal SCCs with a sensitivity of 87.5% and a specificity of 100%. This provides an alternative to p16 immunohistochemical staining of FNA cell block material, which may not be available on all specimens.

Fine-needle aspiration (FNA) biopsy of Hurthle cell containing thyroid nodules can be challenging because of a broad differential that contains both benign and malignant entities. Most nodules diagnosed by FNA as a follicular neoplasm, Hurthle cell type, are benign. This results in unnecessary surgeries and the risk of complications that accompany it. Numerous studies have tried to determine the most useful cytomorphologic features to best make the distinction between benign and malignantHurthle cell nodules. While there are reproducible cytomorphologic features that can favor one over the other, there does not appear to be a single cytologic feature that can entirely exclude a neoplasm. While morphologically Hurthle cell carcinoma is considered a variant of follicular carcinoma, there is evidence to support it may be a distinct entity. In the future, molecular studies might be able to complement the cytomorphologic findings of FNA to provide a more refined and specific diagnosis for Hurthle cell containing thyroid nodules.

BACKGROUND:Endoscopic ultrasonography (EUS) is commonly used in the evaluation of pancreas masses, and when a liver lesion is visualized, it can undergo a fine-needle aspiration (FNA). This can provide diagnostic and staging information. The purpose of the study was to correlate the findings of patients who underwent EUS FNA biopsy of a pancreas lesion and a liver lesion during the same procedure.MATERIALS AND METHODS:The pathology database at Washington University Medical Center was searched for EUS FNA biopsy cases where biopsy of both the pancreas and liver were performed over a consecutive 10-year period (2003-2013). All pathology reports were reviewed, and clinical information and diagnostic results were recorded.RESULTS:A total of 102 cases were identified. For pancreas cases, 79.4% were malignant and for liver cases, 58.8% were malignant. In pancreas lesions categorized as suspicious for malignancy (9%), the liver biopsy provided a diagnosis of malignancy in 67% of cases. A malignant pancreatic cohort demonstrated a 62.9% liver malignancy. A malignant liver cohort corresponded to a malignant pancreas diagnosis in 86.6% of cases and a suspicious-malignant group of 98.3%.CONCLUSIONS:The 102 cases with concomitant EUS FNA biopsy of the pancreas and liver demonstrated the ability to provide a diagnosis of pancreas malignancy and correlate regional metastatic malignancy in the liver. In patients with a pancreas mass and in the appropriate clinical setting, a liver EUS FNA biopsy has the ability to provide a diagnosis of malignancy and demonstrate a high positive predictive value of malignancy in the pancreas (98.3%).