Abstracts From the Pulmonary Pathology Society 2024 Biennial Meeting Open Access

T. Abi-Saab; G. Phenegar; D. Buehler; J. Schulte ([email protected]). Department of Pathology and Laboratory Medicine, The University of Wisconsin School of Medicine and Public Health, Madison.

Context: At our institution, lung transplant biopsies undergo rapid processing, with permanent sections available for same-day read. Owing to flooding, the histology laboratory was temporarily closed, but the frozen section (FS) laboratory remained operational. An FS protocol for lung transplant biopsies was developed and used until histology operations resumed.

Design: During 3 days, 8 lung transplant biopsies were evaluated by using the FS method. Fresh specimens were frozen and sectioned on the cryostat, producing 3 sections 8 µm apart, then hand-stained in hematoxylin-eosin. Preliminary diagnoses were rendered by pathologists with experience reading lung transplant biopsies. Specimens were fixed in formalin for routine processing, with final diagnoses rendered on permanent sections once histology operations resumed.

Results: Five biopsies showed no evidence of rejection (A0B0 or A0BX) and 3 biopsies showed evidence of acute cellular rejection (grades A1B0/A1B1R/A2B0). All biopsies without rejection showed no evidence of rejection on permanent sections. One case with a diagnosis of A1B0 showed A1B1R rejection on permanent sections, a minor discordance. Overall rate of concordance was 88%.

Conclusions: This study highlights the effectiveness of using FS to evaluate lung transplant biopsies for rejection, and the ability to maintain vital surgical pathology services via novel use of the FS. The FS is overall accurate in determining the presence of rejection, but airway-based inflammation may be missed. With the benefits of a reduction in equipment and staff resources, and a more rapid turnaround time, using FS in lung transplant could positively impact the pathology laboratory and patient care.

K. Steinestel¹ ([email protected]); D. Gagiannis²; M. Grunert.^{3 1}Department of Pathology, ²Clinic of Internal Medicine/Pulmonology, and ³Department of Nuclear Medicine, Bundeswehrkrankenhaus Ulm, Ulm, Germany.

Context: Fibroblast activation protein (FAP), a 97-kDa type II transmembrane serine protease, is expressed on reactive stromal fibroblasts at the invasion front of epithelial cancers and in granulation tissue of wound healing. Radiolabeled PET tracers that act as fibroblast-activation-protein inhibitors (FAPis) have been proposed as molecular imaging tools for the visualization of fibrotic remodeling in the lung. Correlation between FAPi-PET/CT and tissue expression of FAP has so far only been demonstrated in very few cases, and FAP expression in other pulmonary lesions with fibromyxoid remodeling has not yet been assessed.

Design: We assessed FAP expression in 25 tissue samples of acute lung injury/interstitial lung disease (organizing diffuse alveolar damage [DAD], organizing pneumonia, and usual interstitial pneumonia/idiopathic pulmonary fibrosis), using immunohistochemistry on transbronchial biopsies or autopsy samples. In 15 cases, molecular imaging data were obtained (68Ga-FAPi-PET/CT). Results were correlated with lung function tests and clinical outcome.

Results: FAP expression can be specifically demonstrated in areas of fibromyxoid tissue remodeling (organizing DAD, fibroblast plugs, and fibroblast foci) and correlates with 68Ga-FAPi-PET/CT activity, restrictive change, and poor clinical outcome (Figure 1).

Conclusions: FAP immunohistochemistry correlates with the results from molecular imaging and highlights areas of active fibrotic remodeling in the lung. We will continue our research to assess whether FAP expression can be validated as a prognostic biomarker for patient stratification in fibrotic interstitial lung disease.

S. S. Batah¹; A. J. Rodríguez-Herrera¹; M. J. F. Marco¹; W. M. Telini¹; A. P. Leme²; R. R. Domingues²; R. F. Carvalho³; A. P. Schnepper³; A. A. Cetlin⁴; D. T. Wada⁵; M. K. Santos⁶; T. R. Nadai⁶; J. A. Baddini-Martinez⁷; V. L. Capelozzi⁸; A. T. Fabro¹ ([email protected]). ¹Department of Pathology and Legal Medicine, Ribeirão Preto Medical School, University of São Paulo, Brazil; ²LNBio, Brazilian Center for Research in Energy and Materials (CNPEM), Brazilian Biosciences National Laboratory, Campinas, SP, Brazil; ³Institute of Biosciences, São Paulo State University, Botucatu, Brazil; ⁴Pulmonary Division, Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo, Brazil; ⁵Department of Medical Images, Hematology and Oncology, Ribeirão Preto Medical School, University of São Paulo, Brazil; ⁶Bauru Medical School, University of São Paulo, Brazil; ⁷São Paulo Medical School, Federal University of Sao Paulo, Brazil; ⁸Department of Pathology, Faculty of Medicine, University of São Paulo, Brazil.

Context: Capelozzi necrosis (CN) is an intraluminal, amorphous, and basophilic substance similar to the peptic necrosis found in gastric ulcers, and it has been associated with microaspiration in fibrosing interstitial lung diseases (f-ILDs) related to systemic sclerosis. Since it has been observed in other f-ILDs, our aim was to investigate the presence of food molecules within it.

Design: Twenty-nine lung surgical biopsies with an alternative diagnosis for usual interstitial pneumonia, and a definitive diagnosis of f-ILD after MDD by American Thoracic Society/European Respiratory Society (ATS/ERS) criteria, were selected between 2017 and 2021. Detailed histologic analysis, proteomics, and digital PCR were performed.

Results: The average patient age was 63 years and 38% were male. Exposure history was present in 86%, and 62% were previously diagnosed with gastroesophageal reflux disease. The intrabronchiolar lumen content consisted of mucus in 88% of cases and CN in 66%. Histologic findings showed epithelial ulceration and adaptive response related to CN. Proteomic analysis of the intrabronchiolar content (CN or mucus) identified 21 peptides (≥24 amino acid sequences) from Bos taurus and Gallus gallus distributed as: 0 mucus⁺/CN⁻; 2 CN⁺/mucus⁻; and 15 CN⁺/mucus⁺. Albumin, vimentin, and inter-α-trypsin inhibitor heavy-chain H2 showed 3 different specific peptides for B taurus. Digital PCR for these genes confirmed the presence of vimentin in 3 samples (0.11 cp/μL each).

Conclusions: Proteomic analysis provides compelling evidence that a portion of CN’s intraluminal content originates from food. Our findings were corroborated by digital PCR. CN could emerge as a novel and dependable histologic biomarker for microaspiration related to interstitial fibrosis.

This work was funded by São Paulo Research Foundation (FAPESP) process numbers 23/10184-3, 23/04199-8, 23/10186-6, 22/02821-0, 19/01517-3; and National Council for Scientific and Technological Development (CNPq) process number 310415/2021-7.

C. Ariño-Palao¹ ([email protected]); L. Peferoen²; T. Radonic.^{2 1}Department of Pathology, Ramón y Cajal University Hospital, Madrid, Spain; ²Department of Pathology, Amsterdam University Medical Center, VU University Amsterdam, Cancer Center Amsterdam, the Netherlands.

Squamous cell papillomas have been associated with low-risk (LR) human papillomavirus (HPV) in the upper respiratory tract and may occasionally be associated with the development of squamous cell carcinoma. They rarely involve the bronchi and lungs. We present a case of a 65-year-old man with a previous LR HPV-11–positive locally aggressive inverted papilloma with destruction of the ethmoidal sinus and orbit. He presented 1.5 years later with multiple bilateral lung lesions in the left and right lungs (Figure 2, A). Histology was available from (1) sinonasal inverted papilloma (Figure 2, B); (2) needle biopsy of the right lung tumor before chemoinduction (Figure 2, C); (3) bronchial biopsy of the left lung mass; and (4) right lower lobectomy after chemoinduction (Figure 2, D). Lobectomy revealed a bronchial papilloma with viral features, a small area of inverted papilloma–like growth, and a complete response of the invasive squamous cell carcinoma found in the pre-chemoinduction biopsy. HPV-PCR of the bronchial papilloma revealed LR HPV-11, and next-generation sequencing unveiled a splice mutation in the TP53 gene in addition to a pathogenic mutation in the TERT promoter gene. Bronchial biopsy of the left lung showed papillomatosis and squamous metaplasia without prominent dysplasia. LR-HPV PCR was performed on the left bronchial biopsy as well as next-generation sequencing on the sinonasal locally aggressive inverted papilloma. Thus, we present a case of histologically proven generalized LR HPV-11–induced papillomatosis of the respiratory tract with risk for locally invasive behavior and development of squamous cell carcinoma.

C. Panayi ([email protected]); K. Giaslakiotis; L. Edmunds; M. Sheaff. Department of Histopathology, Royal London Hospital, Barts Health NHS Trust, London, United Kingdom.

The current landscape of primary SMARCA4-deficient tumors of the thorax includes SMARCA4-deficient conventional non–small cell lung carcinoma and thoracic SMARCA4-deficient undifferentiated tumor (SMARCA4-UT). The differential diagnosis of SMARCA4-UT includes neuroendocrine carcinoma (NEC). We present a case of a thoracic SMARCA4-UT, which was notable for demonstrating both morphologic and immunophenotypic features of NEC. Diagnostic modalities included conventional immunohistology and consensus/expert review. A 36-year-old man presented with chest pain and was found to have a large thoracic mass centered in the mediastinum. Core biopsy revealed a high-grade tumor with necrosis and Azzopardi effect, comprising small round cells with frequent mitotic activity. The morphology strongly resembled small cell lung carcinoma, though occasional nucleoli were appreciated (Figure 3, A). Immunohistochemistry demonstrated diffuse expression of TTF1, synaptophysin, and CD56, whereas SMARCA4 was completely lost in tumor cells (Figure 3, B through D). Pancytokeratin showed only very focal dotlike perinuclear pan-cytokeratin staining. Chromogranin and CD34 were negative. A consensus diagnosis of thoracic SMARCA4-UT was reported. This case demonstrates the extent to which SMARCA4-UT may mimic NEC, including small cell lung carcinoma. SMARCA4 mutations are among the more common alterations recognized in non–small cell lung carcinoma, whereas SMARCA4 deficiency has not been well described in small cell carcinoma. While SMARCA4-UT has a worse prognosis than SMARCA4-deficient non–small cell lung carcinoma, the finding of SMARCA4 deficiency in either setting appears to confer resistance to conventional cytotoxic therapy. Identifying this molecular hallmark therefore has prognostic and therapeutic relevance, and novel management strategies including immune checkpoint inhibition may prove beneficial.

Q. Zhang; K. Sharma; C. Bodnar; J. Schulte ([email protected]). Department of Pathology and Laboratory Medicine, The University of Wisconsin School of Medicine and Public Health, Madison.

Context: Intraoperative frozen section diagnosis is critical in thoracic diseases evaluation. Our study aims to investigate the discrepancy between intraoperative frozen section and permanent section diagnosis in thoracic diseases.

Design: A total of 536 thoracic frozen sections were performed between 2018 and 2021 that were retrospectively reviewed. The discrepancy rate between frozen and permanent sections was calculated.

Results: Of 273 lung samples, 13 cases showed discrepancies between frozen and permanent sections; 3 showed major discrepancy (discrepancy index = 2.92%). Of 189 cases of lymph node samples, 11 cases showed discrepancies; 3 showed major discrepancy (discrepancy index = 3.7%). Of 74 mediastinal or other site samples, 11 showed discrepant results; 8 showed major discrepancy (discrepancy index = 12.84%) (Table). The leading reason for discrepancy at frozen section was undersampled tumor (lung), nonspecific cancer diagnosis (lymph node), and incorrect diagnosis (mediastinum/other sites).

Conclusions: Analysis of the data reveals varying levels of discrepancy across anatomic compartments of the thorax. While lung and lymph node samples show moderate levels of disagreement, mediastinal and diseases at other sites exhibit a considerably higher level of major discrepancy. Major discrepancy in lung samples were likely a result of sampling error. For lymph nodes, the most frequent reason for major discrepancy appears to be incorrect specific diagnosis. The most common reason for discrepant results in the mediastinum or other sites was incorrect diagnosis on frozen section. These findings indicate the necessity for a comprehensive review of diagnostic procedures in the thorax, which may enhance the accuracy and consistency of intraoperative evaluations.

T. Losmanova ([email protected]); F. Hagmann; J. Schäfer; J. Cordey; C. Bückelmann; S. Reinhard; M. Trippel; I. Zlobec. Institute of Tissue Medicine and Pathology, University of Bern, Switzerland.

Context: Bronchoalveolar lavage (BAL) brings an important contribution in diagnosing pulmonary diseases, particularly interstitial lung diseases and alveolar hemorrhage, through its minimally invasive procedure. The analysis of standard cell distribution and the assessment of iron-laden macrophages (the Golde score) are integral to standard medical reports. However, the traditional cytologic method of manual cell counting is subject to interobserver variability and staining quality issues.

Design: To address these issues, we set out to train artificial intelligence (AI)–based algorithms to enhance the accuracy of differential cell counts and Golde score evaluations in BAL samples. We compiled a representative sample cohort from our internal house archive encompassing Hemacolor, Papanicolaou, and iron-stained smears. This was followed by a process of training and internal validation.

Results: The training was conducted using HALO AI software based on whole cell segmentation and subsequent phenotyping selected cells into specific subgroups. In our feasibility validation study, we examined 10 conventional, Hemacolor-stained smears (Figure 4). The validation against manual counts and the data from LIS revealed that the AI-based values on air-dried smears aligned closely with manual methods, exhibiting less than a 5% discrepancy in 10/10, 9/10, and 8/10 smears for eosinophils, lymphocytes and neutrophils, and macrophages, respectively. For Papanicolaou and iron stains, validation is pending.

Conclusions: Given that conventional smears are still prevalent in cytology, they represent a viable material for the application of AI technologies. Moving forward, we plan to conduct further validations with an expanded sample size to integrate this approach into everyday practice to improve the precision and efficiency of BAL diagnostics.

E. Chou; Z. Mahdi; J. Jagirdar ([email protected]). Department of Pathology, Emory University, Atlanta, Georgia.

Hyalinizing clear cell carcinoma (pHCCC) is the latest addition to pulmonary “salivary gland–type neoplasms” in the World Health Organization Classification of Tumours with a handful of published cases currently. It is defined as “a low-grade malignant epithelial tumor with cords, trabeculae, and nests of clear and eosinophilic cells infiltrating within a background of myxohyaline cellular fibrous stroma.” The tumor characteristics and behavior are evolving. We present an endobronchial pHCCC with a smaller mucoepidermoid-like component that transitions into a larger clear cell component. H&E, mucicarmine, and immunohistochemical stains (CK5, CK7, P40, TTF-1, Napsin A, ALK, synaptophysin, Ki-67) were analyzed. Morphologically distinct areas were circled separately for fluorescence in situ hybridization (FISH) analysis using EWSR1 and MAML2 break-apart probes. We present the case of a nonsmoking 77-year-old woman with a 3.9-cm endobronchial lung lesion that microscopically showed distinct low-grade mucoepidermoid and pHCCC morphology (Figure 5, A through H). The tumor focally demonstrated high-grade nuclear features, necrosis, and mitoses. The pHCCC and transitional areas displayed increased mitotic count, compared to the smaller mucoepidermoid area. The tumor extensively involved the bronchial cartilage, perineurium, and a peribronchial lymph node. The tumor was negative for TTF-1 and Napsin A expression, and was diffusely positive for CK5, CK7, and P40 (all 3⁺). Extracellular and cytoplasmic mucin was seen in the glandular component (mucicarmine positive). FISH analysis showed translocations involving the EWSR1 gene in both areas; MAML2 gene rearrangement was not detected in either area. This was a case of a pHCCC with 2 distinct morphologies, both harboring a common EWSR1 translocation. The tumor displays aggressive features in this typically low-grade entity.

J. Kashima; Y. Yatabe ([email protected]). Department of Diagnostic Pathology, National Cancer Center Hospital, Tokyo, Japan.

A 30-year-old woman with von Hippel–Lindau (VHL) disease, who had a history of cerebral hemangioblastoma at age 22 years and renal clear cell carcinoma at age 29 years, had multiple small nodules in the bilateral lungs on follow-up computed tomography imaging (Figure 6, A, arrows). A bronchoscopic biopsy of the 7-mm nodule was performed to exclude metastasis of clear cell carcinoma. The nodule was indeed a cystic lesion, as yellowish fluid was mostly aspirated, and only small tissue fragments were obtained. Histologically, the specimens showed multiple small cystic lesions along normal bronchioles (Figure 6, B). The cysts were lined with 2 layers of cells: CK7⁺ (Figure 6, C), TTF-1⁺, and PAX8⁻ epithelium on the luminal side, and basal CK7⁻ and ERG⁺ (Figure 6, D) vascular endothelial cells. There was no cellular atypia in either component. Pulmonary cystic lesions in VHL have been described previously in a case report with histopathologic features identical to those of our patient. These pulmonary lesions may be the pulmonary counterparts of cystic lesions in other organs observed in patients with VHL disease.

S. S. Batah¹; A. J. Rodríguez-Herrera¹; M. J. F. do Marco¹; M. M. L. Moraes¹; W. M. Telini¹; A. A. Cetlin²; D. T. Wada³; M. K. Santos⁴; T. R. Nadai⁴; A. T. Fabro¹ ([email protected]). ¹Department of Pathology and Legal Medicine, Ribeirão Preto Medical School, University of São Paulo, Brazil; ²Pulmonary Division, Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo, Brazil; ³Department of Medical Images, Hematology and Oncology, Ribeirão Preto Medical School, University of São Paulo, Brazil; ⁴Bauru Medical School, University of São Paulo, Brazil.

Context: The airway-centered interstitial fibrosis (ACIF) is an interstitial lung disease with peribronchial and/or peribronchiolar remodeling, including etiologies as hypersensitivity pneumonitis (HP), microaspiration-related interstitial fibrosis (MARIF), and collagen vascular disease (COL). Our aim was to analyze dendritic cell (DC) profiles in patients with airway-centered interstitial fibrosis as potential biomarkers.

Design: Twenty-one surgical lung biopsies with definitive diagnosis for airway-centered interstitial fibrosis related to HP, MARIF, and COL after MDD by American Thoracic Society/European Respiratory Society (ATS/ERS) criteria were selected between 2017 and 2021. Their clinical data were collected for survival. Immunohistochemistry for mannose, CD1c, Langerin, and CLEC9a with dendritic cell morphology and their morphometry were performed.

Results: Comparative morphometry analysis revealed higher mannose⁺DC in MARIF, CD1c⁺DC, and CLEC9a⁺DC in HP, and Lagerin⁺DC in COL. Additionally, the 21 patients with airway-centered interstitial fibrosis with high expression of CLEC9a⁺DC (>1,50 cells/cm²) presented worse survival, while high expression of CD1c⁺DC (≥2,04 cells/cm²) was associated with better survival.

Conclusions: The immune pathway is critically involved in MARIF, HP, and COL etiologies for ACIF progression and DC is a key cell. Our study demonstrated that different DC immunophenotypes may be a potential biomarker in predicting differential diagnosis and patient survival in ACIF.

This work was funded by São Paulo Research Foundation (FAPESP) process numbers 23/10184-3, 23/04199-8, 23/10186-6, 22/02821-0, 21/09024-6, 19/01517-3; and National Council for Scientific and Technological Development (CNPq) process number 310415/2021-7.

S. S. Batah¹; A. J. Rodríguez Herrera¹; M. J. F. do Marco¹; A. P. Schnepper²; R. F. Carvalho²; J. A. Baddini-Martinez³; A. T. Fabro¹ ([email protected]). ¹Department of Pathology and Legal Medicine, Ribeirão Preto Medical School, University of São Paulo, Brazil; ²Institute of Biosciences, São Paulo State University, Botucatu, Brazil; ³São Paulo Medical School, Federal University of Sao Paulo, Brazil.

Context: A bronchiolocentric pattern of lung injury can be observed in a wide array of conditions, such as microaspiration-related interstitial fibrosis (MARIF) and fibrotic hypersensitivity pneumonitis (f-HP). Although clinical, radiologic, and pathologic findings may resemble each other across many etiologies, early identification of specific pathways of lung injury, based on their molecular and pathologic basis, could aid in a more accurate diagnosis. Hence, our aim was to explore potential translational diagnostic biomarkers at the molecular level.

Design: Nine surgical lung biopsies with definitive fibrosing interstitial lung disease diagnosis for MARIF and f-HP, after multidisciplinary discussion by American Thoracic Society/European Respiratory Society criteria, were selected between 2017 and 2021. Histologic feature and whole transcriptome analyses were performed.

Results: Eighty percent of patients with MARIF were male, while all patients with f-HP were female. Transcriptome analysis revealed 44 genes differentially expressed (padj <.05). MARIF up-expressed genes were related to Y chromosomal genes (DDX3Y, KDM5D, UTY), TGF-β-signaling pathway (YTHDC1), cell stress (HSPA1A), and fibrotic conditions (DDR1), while f-HP up-expressed genes were associated with DNA repair (BRCA2 and CTDP1) and immune response (HLA-DRA, Nanog, and LINC00504).

Conclusions: Despite histologic similarities between MARIF and f-HP, their pathophysiology and transcriptomic profiles diverge significantly. MARIF appears to involve a higher expression of genes associated with the Y chromosome and a stronger fibrotic component, whereas f-HP leans toward inflammatory and immunologic mechanisms. Further investigations are warranted to elucidate and correlate these potential molecular markers with the pathophysiologic pathways of these conditions.

This work was funded by São Paulo Research Foundation (FAPESP) process numbers 23/10184-3, 23/04199-8, 23/10186-6, 22/02821-0, 19/01517-3; and National Council for Scientific and Technological Development (CNPq) process number 310415/2021-7, and FAEPA 639/2023.

G. Phenegar; E. Laumer; M. Stenerson; J. Schulte ([email protected]). Department of Pathology and Laboratory Medicine, The University of Wisconsin School of Medicine and Public Health.

Context: During lung transplant, it is routine to submit explanted lungs for gross and microscopic evaluation. It is less common for evaluation of the donor lungs to be performed alongside the explanted organs. Our pathology department occasionally receives wedge resections of donor lungs along with explants, and these donor lung wedges are processed for gross and microscopic examination. We aim to describe the pathologic processes present in these donor lungs at the time of transplant.

Design: During a 4-year span, 20 explanted lungs had accompanying donor lung wedges submitted to surgical pathology. These lung wedges were formalin fixed, paraffin embedded, sectioned, and stained with hematoxylin-eosin alongside the explanted specimens. Final diagnoses were rendered by a fellowship-trained thoracic surgical pathologist.

Results: All 20 of the donor wedges were grossly normal but revealed histopathologic changes on microscopic evaluation; many wedges showed multiple pathologies. Seven specimens (35%) showed thromboemboli in small or medium pulmonary arteries, 4 (20%) showed chronic bronchiolitis, 4 (20%) showed anthracosis, 3 (15%) showed acute bronchopneumonia, 2 (10%) showed organizing pneumonia, and 1 specimen (5%) was identified with the following: respiratory bronchiolitis, smoker’s macrophages, pulmonary bleb, emphysema, nonspecific chronic inflammation, and acute necrotizing pneumonia.

Conclusions: Our results shed light on the frequency of underlying pathology within transplanted donor lungs. The impact of these findings on subsequent episodes of rejection and graft outcomes is unknown. Because gross examination of lung tissue often fails to detect these pathologies, histologic analysis of lungs prior to transplant may be informative in select scenarios.

K. Nabeshima¹ ([email protected]); T. Furukawa¹; K. Takizawa¹; S. Matsumoto²; M. Hamasaki.^{2 1}Department of Diagnostic Pathology, Fukuoka Tokushukai Hospital, Fukuoka, Japan; ²Department of Pathology, Fukuoka University Hospital and School of Medicine, Fukuoka, Japan.

Context: Discriminating pleural mesothelioma (PM) from reactive mesothelial proliferations (RMPs) can be challenging. In such cases, ancillary assays including immunohistochemistry (IHC)–detected BAP1 or MTAP loss and FISH-detected CDKN2A homozygous deletion are useful. Recently, Chapel et al showed that IHC-detected loss of Merlin, the protein product of the NF2 gene that is frequently altered in mesotheliomas, also effectively distinguished PM from RMPs. Thus, we examined Merlin IHC in our PM cases.

Design: Merlin IHC was performed on 67 PMs, including 47 tissues and 20 cell blocks, and 29 RMP cases, using clone D3S3W of rabbit monoclonal antibody and Ventana BenchMark.

Results: In RMPs, Merlin was expressed in cell membranes and occasionally in cytoplasm, with no loss. In PMs, Merlin expression could not be determined because cells stained heterogeneously in 13% of cases. Merlin loss was found in 49% of PM tissues. In discriminating PMs from RMPs, a dual combination of BAP1 and MTAP IHC yielded an increased sensitivity of 77% compared with their single use, which was further enhanced to 93% by the addition of Merlin IHC. In cases where both tissue biopsy and cell blocks were available, their results matched each other in only 50% of cases, which was quite low, compared to the previous studies on BAP1 IHC and CDKN2A FISH with 100% matches.

Conclusions: Our observations strongly support the data of Chapel et al, suggesting the effectiveness of Merlin IHC in differentiating between PMs and RMPs. However, further examination of heterogeneous Merlin expression in PM tissues and cell blocks is necessary.

S. S. Batah¹; A. J. Rodriguez-Herrera¹; M. J. F. do Marco¹; J. R. S. Chiappetto²; M. Gatto²; S. A. do Vale²; R. A. Prudente²; A. P. Schnepper³; R. F. Carvalho³; J. P. F. Almeida⁴; T. R. Nadai⁵; M. K. Santos⁵; L. S. Wada⁶; J. A. Baddini-Martinez⁷; D. T. Wada⁸; A. A. Cetlin⁹; V. L. Capelozzi¹⁰; B. G. Baldi¹¹; S. Tanni²; A. T. Fabro¹ ([email protected]). ¹Department of Pathology and Legal Medicine, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil; ²Department of Internal Medicine, Botucatu Medical School, Paulista State University, Botucatu, Brazil; ³Institute of Biosciences, Paulista State University, Botucatu, Brazil; ⁴Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Brazil; ⁵Bauru Medical Course, University of Sao Paulo, Brazil; ⁶Department of Surgery and Anatomy, Ribeirão Preto Medical School, University of São Paulo, Brazil; ⁷Paulista Medical School, Federal University of São Paulo, Brazil; ⁸Department of Medical Images, Hematology and Oncology, Ribeirão Preto Medical School, University of São Paulo, Brazil; ⁹Pulmonary Division, Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo, Brazil; ¹⁰Pathology, Faculty of Medicine, University of São Paulo, Brazil; ¹¹Division of Pulmonology, Heart Institute – InCor – Clinical Hospital, Faculty of Medicine, University of São Paulo, São Paulo, Brazil.

Context: Post–COVID-19 pulmonary fibrosis (CPF) is a complication diagnosed by clinical setting and pulmonary function tests or high-resolution computed tomography. It may develop after the organizing diffuse alveolar damage (ODAD) phase. The transcriptomic profile of these patients might resemble other fibrosing interstitial lung diseases (f-ILDs). Our aim was to identify potential biomarkers within the gene expression that indicate fibrosis progression.

Design: We performed RNA-Seq of samples from 4 distinct groups: ODAD, CPF, f-ILD, and control (CTR). In situ confirmation of MUC4, KRT5, MUC5ac, and WNT10a was performed by immunohistochemistry.

Results: The age of patients ranged from 58 to 70 years, with women predominating in the ODAD and f-ILD groups. The CPF group experienced pulmonary complications 324 days after swab that was positive for SARS-CoV-2. At the transcriptomic level, 86 fibrosis-related genes commonly expressed in CPF and f-ILD were identified. Genes such as MUC5ac and WNT10a might play a role in maintaining fibrosis even in the absence of ongoing lung injury, which was confirmed in situ with higher expression in goblet cell hyperplasia and squamous cell metaplasia. In addition, 31 progression-related genes commonly expressed among the 3 groups were identified. Their protein expression was confirmed in situ: MUC4⁺ bronchial goblet cell hyperplasia, and KRT5⁺ basal cell hyperplasia.

Conclusions: For the first time, to our knowledge, our findings have identified a gene expression profile potentially linked to the progression of COVID-19 to pulmonary fibrosis, uncovering shared pathways with f-ILD. Among the progression-related genes, KRT5 and MUC4 stand out and might serve as biomarkers for the early diagnosis of fibrosis progression in transbronchial biopsy samples.

This work was funded by São Paulo Research Foundation (FAPESP) process numbers 23/10184-3, 23/04199-8, 23/10186-6, 22/02821-0, 21/09024-6, 19/01517-3; and National Council for Scientific and Technological Development (CNPq) process number q310415/2021-7.

Federica Pezzuto¹ ([email protected]); Lauren D’Sa²; Nicol Bernardinello¹; Francesca Boscaro¹; Anamaria Chelu³; Cecilia De Chellis¹; Giulia Bacchin¹; Luca Vedovelli¹; Chiara Giraudo¹; Paolo Spagnolo¹; Elisabetta Balestro¹; Fiorella Calabrese.^{1 1}Department of Cardiac, Thoracic, Vascular Sciences and Public Health, University of Padova, Padova, Italy; ²Department of Histopathology, Royal Brompton and Harefield Hospitals, Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom; ³Unità Operativa Pneumologia - Arco - Arco di Trento, Italy.

Context: Progressive fibrotic interstitial lung disease (PF-ILD) poses a challenge in clinical practice, with reliable predictors of its progression remaining elusive. This study aimed to elucidate potential histologic, molecular, radiologic, and clinical markers predictive of a progressive phenotype in patients with fibrotic ILD.

Design: Forty-eight patients with ILD, other than idiopathic pulmonary fibrosis and connective tissue disease–ILD, were enrolled between January 2016 and May 2023. Progression was defined according to the recent guidelines. Clinical, radiologic, and functional data were retrospectively collected. Digitalized whole slide images (hematoxylin-eosin and Masson trichrome) were scanned and blindly assessed with QuPath software. Lymphoid aggregates, follicles with germinal centers, granulomas, fibroblastic foci, and inflammatory cells were quantified (per mm²). Inflammation and fibrosis extent (focal, patchy, extensive) and distribution patterns (interstitial, airway-centered, subpleural) were evaluated. Organizing pneumonia was further quantified (%). Pigmented intra-alveolar macrophages were documented. After RNA extraction, the human Cytokine Network 96-well Plate, including 28 assays targeting cytokine/chemokine network-associated genes, was performed.

Results: Fifteen patients had progression to PF-ILD. This phenotype exhibited lower overall survival (P = .02), a higher prevalence of bronchiectasis, and lower functional parameters at diagnosis than did nonprogressors. A significant overexpression of IL-9 in PF-ILD was observed (P = .049). The Boruta algorithm identified that number of lymphoid aggregates and traction bronchiectasis at diagnosis were predictive of progression.

Conclusions: This comprehensive approach enhances the understanding of pathologic mechanisms underlying progression to PF-ILD, offering potential avenues for improved prognostication and therapeutic intervention. Further studies are needed to confirm the predictive role of these morphologic and molecular biomarkers.

I. Sansano¹ ([email protected]); A. Villar²; B. Caicedo³; F. Pilia²; I. Ojanguren²; L. Del Carpio Bellido.^{4 1}Department of Pathology, Hospital Universitari Vall d’Hebron, Barcelona, Catalunya, Spain; ²Department of Pneumology, Hospital Universitari Vall d’Hebron, Barcelona, Catalunya, Spain; ³Department of Pathology, Universidad Javeriana, Bogotá, Colombia; ⁴Department of Radiology, Hospital Universitari Vall d’Hebron, Barcelona, Catalunya, Spain.

Context: Smoking-related interstitial fibrosis (SRIF) is a pathologically defined form of interstitial fibrosis characterized by alveolar septal thickening by eosinophilic, paucicellular fibrosis composed of “ropey-appearing” collagen, usually accompanied by emphysema and pigmented airspace macrophages. The term SRIF was first introduced as a “clinically occult” finding and it has been generally assumed that SRIF is clinically insignificant, but this assumption has been recently challenged.

Design: We identified patients with clinical and imaging evidence of interstitial lung disease (ILD) and transbronchial cryobiopsy specimens with SRIF alone, without any other pathologic findings that could account for clinical ILD.

Results: Four cases met inclusion criteria (Table); all 4 (3 men and 1 woman, aged 55–67 years, with a mean age of 59 years) were current smokers and were evaluated owing to respiratory symptoms and abnormal pulmonary function tests, specifically reduced diffusing capacity for carbon monoxide (DLCO). All 4 patients had bilateral ground-glass opacities, which correlated with histopathologic SRIF in cryobiopsies. Upon follow-up of 1 to 2 years in 3 patients, 1 experienced worsening of dyspnea, and another had a significant decline (>10%) in DLCO. Two patients received bronchodilators, 1 received corticosteroids, and all 4 were advised for smoking cessation (only 1 quit smoking).

Conclusions: As reported previously in surgical lung biopsies, SRIF can occasionally present as clinically meaningful ILD in relatively young heavy smokers, characterized by bilateral ground-glass opacities. Although limited sampling complicates interpretation of clinical significance, a pathologic diagnosis of SRIF can also be made in transbronchial cryobiopsies.

Chhabilal Bastola¹ ([email protected]); Danilo Enriquez¹; Harish Patel¹; Eugene Shostak²; Ambika Kaphle.^{3 1}One Brooklyn Health (Brookdale Hospital Medical Center), New York; ²Weill-Cornell Medical Center, New York; ³Nobel Medical College, Nepal.

IgG4-related disease is well established as a chronic inflammatory condition characterized by fibrosis and dense infiltrates of IgG4 plasma cells. There are wide clinicopathologic manifestations, including in the lung. Here we present an unusual case in which IgG4-related disease mimicked bronchogenic carcinoma and allowed for appropriate treatment. A 66-year-old woman presented with dry cough, hemoptysis, and 10-pound weight loss during the prior 3 months. Computed tomography (CT) of the chest revealed a 4.7 × 2.3 × 6.1-cm right upper lobe spiculated mass with subpleural involvement, suggestive of either neoplasia or pneumonia. Bilateral emphysema was present. A bronchoscopic biopsy was negative for malignancy. Fine-needle aspiration cultures revealed Nocardia versus Actinomyces organisms. Quantiferon, Fungitell, and galactomannan assays, and acid-fast bacilli cultures were negative. The subject subsequently developed a right basal ganglia infarction and was treated with Plavix. A repeated chest CT showed an increase in the right lung mass with cavitations and consolidation to 7.1 × 3.0 cm. Robotic bronchoscopy was performed and it was again determined as negative for malignancy, showing marked acute inflammation. Infection stains were negative. IgG4 immunostain highlighted scattered clusters of IgG4⁺ plasma cells (up to 30 cells/HPF). Immunostains for CD3 and CD20 showed mixed T and B lymphocytes, respectively. κ and λ in situ hybridization showed polytypic plasma cells with no evidence of lymphoma. Total serum IgG levels were elevated with IgG class 1, 4 significantly elevated with total serum IgG 2035 (600–1540 mg/dL). The case was discussed at the tumor board at Cornell University, diagnosed as IgG4-related disease, and the patient was started on steroids with significant clinical improvement. This case highlights the clinical and morphologic diversity of IgG4-related disease in the setting of radiologic mimicry of neoplasia. Accurate diagnosis prevented more significant surgical procedures for neoplasia. IgG4-related disease can be effectively managed, making early diagnosis important.

L. Osgood¹ ([email protected]); R. Attanoos.^{1,2 1}Department of Cellular Pathology, University Hospital of Wales, Cardiff, United Kingdom; ²Cardiff University, Cardiff, United Kingdom.

Context: There is a strong association between pleural mesothelioma and asbestos in men, frequently relating to prior workplace commercial amphibole exposure. In women, the association is less, estimated to be 23%.

Design: This study analyzed pleural mesotheliomas in women that were submitted to a national referral laboratory for mineral fiber analysis. Presence of objective biomarkers (pleural plaques and/or asbestosis) and exposure history were recorded. Transmission electron microscopy with energy dispersive x-ray spectrometry was performed on lung tissue. Asbestos fiber type and counts were recorded and compared with the laboratory control population.

Results: Thirty-three cases were studied. Four (12%) had elevated commercial amphibole asbestos fiber counts; 19 (58%) had detectable asbestos within the background control range; and 10 (30%) had none detected. Pleural plaques were reported in 3 cases (2 with no asbestos fibers detected and 1 within background control range). No asbestosis was reported. None of the subjects had clear occupational exposures. Cases were grouped according to whether they were considered likely asbestos related (elevated fiber counts and/or presence of biomarkers/clear exposure history); likely nonasbestos related (fiber count within background control range and absence of biomarkers/exposure history); and not asbestos related (no asbestos detected and absence of biomarkers/exposure history) (Table).

Conclusions: The attributable asbestos fraction of pleural mesotheliomas in women in this study is comparable to that of analytic epidemiology at 21%. Limitations include small sample size and referral bias of cases, which have equivocal links with asbestos (cases referred from coroners and not medicolegal).

Attanoos provides expert testimony in asbestos litigation for claimants, defendants, and joint basis.

M. J. F. Do Marco; S. S. Batah; A. J. Rodriguez-Herrera; A. T. Fabro ([email protected]). Department of Pathology and Legal Medicine, Ribeirão Preto, São Paulo, Brazil.

Context: Anthracosis is black deposits of carbon due to inhalation of coal dust by repeated inhalation of particulate air pollutants, smoke, or coal dust particle, serving as an index of air pollution exposure. While anthracotic deposition has been associated with environmental pollution, our aim was to determine the deposition pattern in 4 lung compartments from population-based geolocation.

Design: Hematoxylin-eosin–stained slides containing lung tissue samples from 498 autopsies from 3 cities were analyzed for the presence of anthracosis in lung compartments (bronchovascular, septal, intralobular septal, and pleural) and population-based geolocation between 2010 and 2022. The urban environment included industrial city A; sugar cane–producing city B; and city C, rural.

Results: City A had the highest level of anthracosis, followed by cities B and C, respectively. The compartments with the most anthracosis were the bronchovascular and septal compartments. City B had more anthracosis in the bronchovascular compartment, while cities A and C had higher levels in the septum. In green spaces, there was a lower presence of anthracosis.

Conclusions: The lungs of populations in heavily industrialized or agriculturally burned cities retain more anthracosis owing to heightened exposure to pollution, albeit with distinct deposition profiles in pulmonary lymphatic clearance pathways, suggesting varying mechanisms of pulmonary adaptation in the histogenesis of lung lesions.

This work was funded by São Paulo Research Foundation (FAPESP) process numbers 23/10184-3, 23/04199-8, 23/10186-6, 22/02821-0, 21/09024-6, 19/01517-3; and National Council for Scientific and Technological Development (CNPq) process number 310415/2021-7.

A. Velasquez-Evers¹ ([email protected]); J. Overton²; S. Erturk¹; Y. Al Hmada¹; R. Velagapudi.^{1 1}Department of Pathology, University of Mississippi, Jackson; ²School of Medicine, University of Mississippi, Jackson.

Inflammatory myofibroblastic tumor (IMT) is a relatively rare spindle cell neoplasm of unknown etiology, predominantly seen in children and young adults. About 50% to 60% show anaplastic lymphoma tyrosine kinase gene (ALK) rearrangements with multiple fusion partners. We present a case of IMT in a patient with epithelioid clustering in a myxoid background and a novel pathogenic mutation. A 42-year-old woman with obstructive sleep apnea presented with shortness of breath and chest pain. Imaging identified a 2.3-cm endobronchial lesion completely occluding the left main stem bronchus and a surgical debulking was performed. Histopathology showed proliferation of plump spindle cells, arranged in short fascicles with minimal mitotic activity, and intermixed lymphocytes in a myxoid background (Figure 7, A through C). Interestingly, there was focal clustering of epithelioid cells. Initially, a broad differential diagnosis was considered, including low-grade sarcoma, IMT, carcinoid tumor, and poorly differentiated carcinoma. Extensive immunohistochemical workup showed tumor cells were positive focally for desmin and diffusely for ALK (Figure 7, D), thus diagnosing IMT. Molecular testing identified a fusion involving tumor necrosis factor receptor–associated factor 3 (TRAF3) and ALK. To our knowledge, this is the first report in the English literature of this fusion in an adult. One previous report of this fusion was described in a pediatric patient with similar histopathology including focal epithelioid clustering, although ALK immunohistochemistry was negative. Targeted chemotherapy with novel ALK inhibitors has proven to be beneficial with these mutations. However, our patient is currently monitored without chemotherapy as subsequent tumor bed resection was negative.

S. Yeon¹; J. Liu¹; G. Venkataraman²; A. Husain²; V. Ananthanarayanan¹ ([email protected]). ¹Department of Pathology, University of Illinois at Chicago Hospital and Medical Center, Chicago; ²Department of Pathology, University of Chicago Medical Center, Chicago, Illinois.

Context: Malignant mesothelioma (MM) is a rare and challenging malignancy with complex pathogenesis and limited prognostic biomarkers beyond histologic subtypes. The grading of epithelioid mesothelioma, highlighted by The Cancer Genome Atlas (TCGA) study, has become a crucial prognostic indicator. Our study aims to investigate prognostic biomarkers in the TCGA mesothelioma cohort, with a focus on epithelioid MM grading.

Design: Data from 86 patients in the TCGA cohort were accessed via the UCSC Xena browser. Clinicopathologic parameters, RNA-seq expression, and somatic mutation data were extracted. Epithelioid mesothelioma grading was conducted by using the nuclear grading system based on whole slide image (WSI) from the TCGA database, excluding low-quality images. Of the 56 epithelioid MM cases, 48 were graded and subclassified by mitotic count and presence of necrosis. Statistical analysis was performed with Rstudio and Stat 18.

Results: Baseline cohort data, stratified by histologic subtypes and grading, revealed older patients with nonepithelioid MM had shorter median overall survival (P = .01). A 4-gene expression score (“MESO4”), including ANKRD1, FOXD1, PARP10, and TRIP13, significantly influenced outcomes irrespective of histologic subtype. Within epithelioid MM, MESO4 further stratified subgroups with distinct OS24 rates (74.2% and 31%). Epithelioid MM, which was graded and subdivided by mitotic count, showed a significant association with TRIP13 (P = .005) and ANKRD1 (P = .047). None of the 4 genes showed significant correlation with presence of necrosis (Table).

Conclusions: This analysis identified a 4-gene signature capable of stratifying epithelioid MM within the TCGA cohort, particularly within the epithelioid MM subgroup, offering insights into prognosis.

Christiane Kuempers¹ ([email protected]); Dörte Nitschkowski^2,8; Karina Stein^2,8; Tobias Jagomast³; Carsten Heidel¹; Jutta Kirfel¹; Sven Perner⁴; Martin Reck^5,8; Christian Kugler^6,8; Ole Ammerpohl⁷; Torsten Goldmann.^{2,8 1}Institute of Pathology, University Hospital Schleswig-Holstein, Campus Luebeck, Luebeck, Germany; ²Histology, Research Center Borstel-Leibniz Lung Center, Borstel, Germany; ³Medical Clinic III, Pulmonology, University Hospital Schleswig-Holstein, Campus Luebeck, Luebeck, Germany; ⁴MVZ Center for Outpatient Oncology GmbH, Tuebingen; ⁵Oncology, LungenClinic Grosshansdorf GmbH, Grosshansdorf, Schleswig-Holstein, Germany; ⁶Surgery, LungenClinic Grosshansdorf GmbH, Grosshansdorf, Schleswig-Holstein, Germany; ⁷Institute of Human Genetics, University Medical Center Ulm, Germany; ⁸Airway Research Center North (ARCN), German Center for Lung Research (DZL), Germany.

Context: Therapy for non–small cell lung cancer (NSCLC) has made immense progress in recent years. However, in the context of personalized therapy, there are many more options for pulmonary adenocarcinomas than for squamous cell carcinomas (SCCs). For the latter, valid prognostic markers in addition to common markers such as TNM and grading are also lacking. Serpin B13 belongs to the superfamily of serpins and acts as a serine proteinase inhibitor. Serpin B13 is a cytoplasmic protein that plays a role in the proliferation and differentiation, for example, of keratinocytes. Here, we describe serpin B13 expression at the protein level and its epigenetic modifications and examine the impact on overall survival, using lung SCC tissues.

Design: We analyzed epigenetic modifications of the serpin B13 gene in NSCLC and its expression on RNA-level by transcriptome analysis. The expression of serpin B13 on the protein level was assessed by immunohistochemistry using an independent cohort of 125 patients with SCC and its expression was correlated with overall survival and immune infiltration.

Results: We revealed a significant difference in methylation of serpin B13 loci in NSCLC and showed that mRNA and protein expression of serpin B13 is upregulated in pulmonary SCC. High protein expression of serpin B13 was significantly associated with a better overall survival. Moreover, carcinomas with low immune infiltration showed significantly less protein expression of serpin B13 than carcinomas with high immune infiltration.

Conclusions: Our results suggest serpin B13 to be a novel prognostic biomarker for pulmonary SCC. Its implementation in future routine diagnostic workup could be reasonable.

Marie Maillard¹; Christina Neppl²; Philipp Zens^3,4; Julie Anex¹; Sabina Berezowska¹ ([email protected]). ¹Institute of Pathology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland; ²Institute of Pathology, Heinrich-Heine University and University Hospital of Duesseldorf, Germany; ³Institute of Tissue Medicine and Pathology, University of Bern, Bern, Switzerland; ⁴Graduate School for Health Science, University of Bern, Bern, Switzerland.

Context: Tumor budding (TB) is an established independent prognostic factor in colorectal carcinoma and its importance is increasing in other cancers. Grading lung squamous cell carcinoma (LUSC) is still controversial, and TB is a promising prognostic parameter, making it a candidate for inclusion in tumor-grading schemes. If TB is to become a standard part of the LUSC pathology report, it should be easily performable, clearly defined, and as reproducible as possible.

Design: We aimed to assess interobserver variability evaluating TB in LUSC. Two certified pathologists independently scored 714 slides (preoperative biopsies and corresponding resection specimens from 249 patients) according to the recommendations of the International Tumor Budding Consensus Conference (ITBCC). Divergent cases were rediscussed and reasons for discrepancy were classified.

Results: Moderate Cohen κ scores were reached when individually considering the TB scores of different locations (biopsies: 0.680; peritumoral budding: 0.651; intratumoral budding: 0.416). Discordant cases significantly more often had intermediate or high TB scores (P < .001). Consensus could be reached in all cases. Six causes of discordant TB scoring were identified: morphologic interpretation (44%), choice of hotspot (41%), extensive inflammation (10%), interpretation of hotspot localization (2.5%), and extensive necrosis (1%). Transcription errors caused discrepant results in 6 cases (2.5%).

Conclusions: We report the possibility to reliably score TB according to the ITBCC guidelines in LUSC and disclose the reasons for interobserver variability. Future guidelines should encompass recommendations on how to assess TB in those difficult and equivocal situations highlighted in the study.

T. Bowers¹ ([email protected]); F. Medeiros¹; L. Edmunds¹; A. Nicholson.^{2 1}Cardiothoracic Pathology Department, Essex Cardiothoracic Centre, Basildon, Essex, United Kingdom; ²Department of Histopathology, The Royal Brompton and Harefield Hospitals, London, United Kingdom.

We report the case of a 66-year-old man presenting with a cerebrovascular event in May 2022 with concurrent history of weight loss for 3 months and shortness of breath since 2 years prior. The patient was an ex-smoker, with a 40 pack-year history, and coronary artery bypass grafting 9 years previous. Cross-sectional imaging showed irregular thickening and enhancing of the right pleura with moderate effusion (Figure 8, A). Video-assisted thorascopic surgery (VATS) pleural biopsy was performed, showing fibrotic pleura, lined by atypical squamous epithelium suggestive of malignancy (Figure 8, B), with focal evidence of invasion (Figure 8, C), positively expressing P40 (Figure 8, D) and negative for calretinin. In the absence of lung lesions, and any prior or present malignancy, primary pleural squamous cell carcinoma (PPSCC) was diagnosed. The patient was managed with single-agent pembrolizumab, but the disease progressed with nodal involvement and a fatal outcome 14 months after diagnosis. PPSCC is an exceedingly rare entity, with only a handful of cases described. VATS biopsy performed in this case facilitated the diagnosis of PPSCC, which could have been misdiagnosed as metastatic carcinoma on a core biopsy. Radiologically, features of PPSCC are indistinct of those of malignant mesothelioma. Prior reports highlight the role of chronic inflammation in tumorigenesis, with cases described in patients who experienced traumatization of the thoracic cavity, namely surgical, prolonged pleural drainage, or pulmonary fistulae. Diagnosis of malignancy arose approximately 10 years after trigger events. Treatment modalities include radiotherapy, chemotherapy, and immunotherapy, singly or combined. Greater awareness of this entity is necessary to allow prompt diagnosis and treatment.

B. Caicedo¹; D. Clofent²; K. Loor²; S. Alava³; T. De la Concepción³; M. Sesé³; Y. No Garbarino³; A. Martínez⁴; J. Hernández-Losa³; M. Culebras²; M. Dinarès³; I. Sansano³ ([email protected]). ¹Department of Pathology, Universidad Javeriana, Bogotá, Colombia; ²Department of Pneumology, ³Department of Pathology, ⁴Department of Oncology, Hospital Universitari Vall d’Hebron, Barcelona, Catalunya, Spain.

Context: Obtaining a sample by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is essential for lung cancer diagnosis and staging. Recent advances in neoadjuvant therapy demand molecular testing (next-generation sequencing and/or PD-L1) in operable stages, which means doing more with an already small sample. For this reason, new sample collecting tools, such as histologic needles (like cytologic needles but with a bevel) have been introduced.

Design: We reviewed 50 EBUS-TBNA specimens from mediastinal lymph nodes and pulmonary masses of patients with suspected lung cancer. Diagnosis and suitability for molecular testing from material obtained with 22G cytologic needles and 22G histologic needles were evaluated.

Results: All 50 cytologic samples were diagnostic, while 10 histologic samples (20%) were not representative of the tissue sampled (inadequate). Six biopsies (12%) and 8 cytologies (16%) were negative, 26 (51%) and 33 (66%) showed primary lung carcinoma, while 8 (16%) and 9 (20%) obtained other diagnoses of malignancy. Of the 33 patients with lung cancer, 28 required molecular studies. Both biopsy and cytology were suitable in 10 of 28 cases (36%). In 5 of 28 (18%), neither cytology nor biopsy were enough. In 3 of 28 (11%), the biopsy was not sufficient, and in 9 (32%) the cytology was not. Considering diagnosis and molecular testing together, cytology and biopsy were similar in 25 of 50 EBUS-TBNA specimens (50%), biopsy was superior in 12 (24%), and cytology was superior in 13 (26%).

Conclusions: In this study, cytology and histologic biopsy are complementary—cytology had higher diagnostic yield, with needle biopsy providing additional material for necessary molecular testing. The effectiveness of cryoprobe sampling that allows greater tissue retrieval is being evaluated.

Alex McGeough ([email protected]); Divya Sharma. Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio.

Context: Nodal metastasis has a significant impact on overall survival in lung cancer with significantly better survival seen in N1a versus N1b. Node retrieval relies on intraoperative procurement and dissection techniques at the grossing bench. Most grossing protocols capture stations 10-12 with no published protocols for capturing station 13-14 nodes. We describe a novel technique to improve N1 nodal retrieval and impact on disease stage.

Design: A novel grossing protocol was created for lobectomy or greater lung resections to increase N1 node yield. A demonstration was held to teach pathology assistants and residents the protocol. After 7 months, retrospective analysis was performed on specimens from February 1, 2023, to April 12, 2024, with September 16, 2023, marking the intervention’s start, with information including the number of lymph nodes in each N1 nodal station, metastasis, and pathologic stage. The average was calculated for each station and total lymph nodes and then compared by using t test for independent means.

Results: A total of 92 eligible cases were found: 40 pre intervention and 52 post intervention. The average number of lymph nodes was statistically significantly higher post intervention in stations 11-12, stations 13-14, and total lymph nodes (P < .005). It was not significantly higher in station 10 (Table). The number of cases positive for lymph node metastasis also increased post intervention: 20% and 29% of cases, respectively (25% of cases were upstaged). The number of cases with metastasis identified in station 13-14 nodes doubled from 2 to 4 cases.

Conclusions: Our novel grossing protocol is an effective method for increasing N1 nodal yield in lung resections. In postintervention cases 13/52 were upstaged from pN0 to pN1a or pN1a to pN1b owing to positive station 13-14 nodes.

Erik Thunnissen¹ ([email protected]); Onno Mets²; Ronald Damhuis³; Birgit Lissenberg-Witte⁴; Idris Bahce⁵; Teodora Radonic¹; Chris Dickhoff⁷; Gianluigi Arrigoni⁸; Daniela Finocchiaro⁸; Hans Blaauwgeers.^{6 1}Department of Pathology, Amsterdam UMC, VU University Medical Center, Amsterdam, the Netherlands; ²Department of Radiology and Nuclear Medicine, Amsterdam UMC, VU University Medical Center, VU University, Amsterdam, the Netherlands; ³Department of Research, Netherlands Comprehensive Cancer Organization, Utrecht, The Netherlands; ⁴Department of Epidemiology and Data Science, Amsterdam UMC, VU University Medical Center, VU University, Amsterdam, the Netherlands; ⁵Department of Pulmonary Medicine, Amsterdam UMC, VU University Medical Center, Amsterdam, the Netherlands; ⁶Department of Pathology, OLVG LAB BV, Amsterdam, the Netherlands; ⁷Department of Cardiothoracic Surgery, Amsterdam UMC, VU University Medical Center, Amsterdam, the Netherlands; ⁸Department of Pathology, San Raffaele Scientific Institute, Milan, Italy.

Context: The World Health Organization (WHO) recognizes adenocarcinoma in situ (AIS) for tumors that are up to 3 cm in size. The aim of this study was to examine pulmonary adenocarcinomas larger than 7 cm in diameter to see if any cases fit the criteria of AIS.

Design: In 2 cases with radiology showing a pneumonia-like pattern, the whole lobe was prospectively examined. From the previously reported nationwide retrospective cohort of 683 patients who had undergone lung surgical resection for pT3N0M0 NSCLC, a subset of 112 patients had adenocarcinomas larger than 7 cm.

Results: In 2 prospective examined lobes, the number of embedded tissue blocks was 130 and 300 plus, respectively. The histologic diagnosis supported by additional staining was AIS, both with a size of 16 cm. Criteria for invasion were detected in none of the slides. In the retrospective study, 3 of 112 cases were reclassified as nonmucinous AIS. Radiologic imaging showed a consolidation pattern in these cases, similar to that seen in pneumonia, while the invasive control cases had a primarily solid appearance. Follow-up showed a clinical recurrence-free survival in the patients with AIS.

Conclusions: AIS can be diagnosed in tumors larger than 7 cm. The restriction of 3-cm size criterion for AIS in the current WHO classification of pulmonary adenocarcinomas may be reconsidered.

S. B. Silva¹; A. A. Sedassari¹; O. M. Y. Takiguchi¹; A. J. Rodríguez-Herrera²; S. S. Batah²; A. T. Fabro² ([email protected]). ¹Department of Medical Imaging, Hematology, and Oncology, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil; ²Department of Pathology and Legal Medicine, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil.

Context: The recent classification of small cell lung cancer (SCLC) into gene-expression molecular subtypes underscored the presence of a spectrum of neuroendocrine (NE) phenotypes. Although such phenotypes could be easily identified owing to their lack of immunohistochemical (IHC) markers, whether they have different clinical and pathologic hallmarks remains unclear.

Design: Ninety-five patients diagnosed with SCLC by NE IHC analysis were retrospectively selected. NE-phenotyping status was assessed according to IHC NE expression. Overall and progression-free survival were evaluated according to NE phenotypes. Samples from NE phenotypes were selected after survival analysis for pathologic (necrosis, desmoplasia, hyalinization, tumor-infiltrating lymphocytes) and immunohistochemical (NE markers, TTF-1, Ki-67) analysis.

Results: Thirty patients were considered with an NE-negative phenotype. Overall survival following first-line chemotherapy was improved in those with a double-negative NE phenotype (HR0.4, P = .03; Cox proportional hazards model: HR0.38, P = .01). In the pathologic assessment, NE-positive patients significantly coexpressed chromogranin, synaptophysin, and TTF-1, whereas double-negative patients preserved CD56 expression. In addition, double-negative samples had more necrosis but less desmoplasia and hyalinization. Finally, patients with NE phenotype were not associated with an immune-excluded phenotype, the predominant pattern for NE SCLC.

Conclusions: NE IHC markers may help identify a subgroup of patients with SCLC with specific clinical and pathologic profiles.

This work was funded by São Paulo Research Foundation (FAPESP) process numbers 23/10184-3, 23/04199-8, 23/10186-6, 22/02821-0, 21/09024-6, 19/01517-3; and National Council for Scientific and Technological Development (CNPq) process number 310415/2021-7.

A. A. Hitt ([email protected]); J. M. Carney. Duke University Medical Center, Department of Pathology, Durham, North Carolina.

Barium sulfate is a contrast medium used for the evaluation of dysphagia and other gastrointestinal conditions, commonly known as a “barium swallow study.” A potential risk includes aspiration, which increases with age, in the setting of known oropharyngeal dysphagia, head and neck cancers, and vomiting. While barium is considered relatively inert, aspiration of the media has been associated with hypoxia, respiratory failure, and pneumonia. We describe a case of chronic hemoptysis of unclear etiology in a 71-year-old patient with a history of moderate aspiration identified on a barium swallow study. Additional history was significant for a remote diagnosis of squamous cell carcinoma of the larynx, for which the patient underwent chemotherapy and radiation. A chest computed tomography scan showed ground-glass opacities in the left lower lobe with surrounding calcific hyperdensities (Figure 9, A). A bronchoscopy performed did not demonstrate a clear etiology of the patient’s hemoptysis, and the patient underwent a left lower lobe bisegmentectomy. Gross evaluation of the lung revealed multifocal, chalky consolidations (Figure 9, B). Microscopic examination demonstrated a marked chronic fibroinflammatory infiltrate present in association with abundant refractile/polarizable crystalline material. Abundant intra-alveolar hemosiderin-laden macrophages were also observed, features in keeping with hemoptysis (Figure 9, C and D). The polarizable material identified is characteristic of barium sulfate. Adverse sequelae of aspirated barium sulfate are well documented. While hemoptysis has been described in this setting, the degree and unclear etiology clinically, highlights the diagnostic challenge. Moreover, this case emphasizes the importance of clinical, radiographic, and pathologic correlation in securing a diagnosis.

A. T. Fabro¹ ([email protected]); S. S. Batah¹; A. J. Rodriguez-Herrera¹; C. B. Ribeiro²; K. M. da Costa²; A. W. Gonçalves-Ferri³; L. G. Galli⁴; V. L. Capelozzi⁵; L. Sbragia.^{2 1}Department of Pathology and Legal Medicine, ²Division of Pediatric Surgery, Department of Surgery and Anatomy, ³Department of Pediatrics, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil; ⁴Cardiology Division, Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil; ⁵Department of Pathology, Faculty of Medicine, University of São Paulo, São Paulo, Brazil.

Context: The development of congenital diaphragmatic hernia (CDH) is marked by lung hypoplasia and vascular remodeling, leading to persistent pulmonary hypertension and notable rates of mortality and morbidity. While existing literature has primarily concentrated on the analysis of the “functional” gas-exchange capillaries, our research endeavors to investigate the role of nonfunctional capillaries—those not actively involved in gas exchange—in the pathophysiologic cascade that ultimately concludes the honeymoon period (or ventilatory stability period) in CDH.

Design: Formalin-fixed, paraffin-embedded samples from human CDH autopsy cases were collected. Morphometric analysis of capillary and artery was performed for all cases, using H&E, red picrosirius, and Verhoeff staining.

Results: Morphometric analysis revealed a significant increase in nonfunctional capillaries in human CDH versus normal lungs. Higher capillary density correlated with later mortality. Moreover, significant alterations were observed in the adventitial layer of the pulmonary vasculature, particularly in smaller arteries, characterized by pronounced thickening. These changes were associated with a decline in elastic recoil and wave amplitude, contributing to the progressive deterioration of the PaO2:FiO2 ratio, pulmonary congestion, and ultimately mortality.

Conclusions: To our knowledge, this is the first study to demonstrate that nonfunctional pulmonary capillaries are increasing in human patients. Pulmonary hypertension may be a protective factor in CDH by attempting to prevent excess lung blood circulation in these nonfunctional capillaries, and prolong the period of ventilatory stability. The exhaustion of elastic fibers, loss of elastic recoil, and wave amplitude result in pulmonary congestion, refractory hypovolemic shock, and potentially fatal outcomes.

This work was funded by São Paulo Research Foundation (FAPESP) process numbers 22/02821-0, 23/10186-6, 23/04199-8, 21/09024-6, 22/12021-1; and National Council for Scientific and Technological Development (CNPq) process number 310415/2021-7.

S. Ualiyeva; H. Liao; A. Basu ([email protected]). Department of Pathology and Laboratory Medicine, Tufts Medical Center, Boston, Massachusetts.

Light chain deposition disease (LCDD) is a rare disorder, which consists of <1% of monoclonal gammopathies, and is characterized by monoclonal immunoglobulin light-chain deposition in organs like kidneys, heart, and liver. LCDD rarely affects the lung and is usually associated with an underlying plasma cell dyscrasia or autoimmune process. Pulmonary amyloidosis is commonly characterized by AL-subtype and displays apple-green birefringence with Congo red stain. The aim of the study is to present a case of lung nodule with LCDD/non-congophilic AL amyloidosis. A 57-year-old woman with Sjogren syndrome presented with shortness of breath, concerning for interstitial lung disease in relation to her Sjogren syndrome. She underwent chest computed tomography, which revealed a 1.7-cm lung nodule at the right upper lobe. On repeated scanning, the nodule was increasing in size and positron emission tomography showed metabolic activity with SUV of 7.3. The lesion was biopsied and histologically examined. Liquid chromatography–mass spectrometry (LCMS) was performed on peptides extracted from amorphous extracellular material from the paraffin-embedded specimen. Tissue analysis revealed lung parenchyma with amorphous substance that did not stain with Congo red. LCMS detected predominance of peptides associated with κ immunoglobulin light chains. It also detected amyloid chaperone proteins from non-congophilic material (Apolipoprotein-A-IV, SAP, Apolipoprotein-A-I, Apolipoprotein-E). Hematopathology workup ruled out lymphoproliferative disorder. Both LCDD and amyloidosis may rarely involve the lung, resulting in the deposition of abnormal proteins. LCDD involves the deposition of monoclonal immunoglobulin light chains, commonly seen in Sjogren syndrome, whereas amyloidosis involves the deposition of congophilic amyloid fibrils. We report an unusual case of immunoglobulin-associated process lying between κ light chain deposition disease and non-congophilic AL (κ)-type amyloidosis (including with and without polarization; Figure 10, A through D).

J. L. Griffin^1,2; S. Higginson¹; H. L. Crane^1,2; K. Hoyles¹; G. Dammery-Quigley¹; L. Brammar¹; K. L. Lloyd^1,2 ([email protected]). ¹Department of Histopathology, Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, United Kingdom; ²University of Sheffield, Sheffield, United Kingdom.

Context: Molecular testing for actionable mutations is an essential aspect of lung cancer care. Rapid turnaround of molecular results by the pathology laboratory is important so patients can begin targeted therapies promptly. We implemented a testing strategy using the Idylla (Biocartis) multiplex PCR platform. Here we present our experience of this approach and its effects on turnaround times (TATs), compared to next-generation sequencing (NGS) and immunohistochemistry (IHC).

Design: Consecutive patients with non–small cell lung cancer during a 3-year period were identified from our laboratory information management system. Clinicopathologic and TAT data were retrieved and analyzed.

Results: In total, 1300 patients underwent molecular testing. We report an interim analysis of 450 patients. The cohort was 51% female, median age of 72 years, with stage 4 disease accounting for 37% of the cohort. Resections, endoscopic ultrasound-guided biopsies, and radiology-guided core biopsies comprised 36%, 21%, and 20% of samples, respectively. In all 762 tests were done with the Idylla platform (EGFR: n = 335, KRAS: n = 140, BRAF: n = 145, fusion panel: n = 142). A total of 12% of patients had EGFR mutations. KRAS and BRAF mutations were found in 20% and 1% of patients, respectively. Testing with the Idylla fusion panel found structural variants in 7% of patients. The fusion panel TAT was faster than for ALK and ROS immunohistochemistry (median, 7 versus 15 days; P < .001). Paired NGS and Idylla testing (n = 32) showed concordant results and faster TAT for Idylla (22 versus 7 days; P < .01).

Conclusions: The Idylla platform provides faster TATs than NGS and IHC for actionable mutations.

H. Shirsat¹ ([email protected]); Meunier Danielle¹; Adam Bassili.^{2 1}Department of Pathology, ²Department of Thoracic Surgery, Royal Jubilee Hospital, Victoria, British Columbia, Canada.

A 79-year-old man with a growing right middle lobe lesion, consistent with carcinoma on positron emission tomography–computed tomography (PET-CT), underwent a wedge resection. Gross examination showed a solid white peribronchiolar lesion measuring 1.1 cm. On histology, the lesion was well demarcated and composed of monomorphic large cells with abundant eosinophilic cytoplasm featuring unusual striations. A lymphoplasmacytic infiltrate was admixed with the large cells. Immunohistochemical stains for oncocytic carcinoid (synaptophysin, chromogranin), metastatic carcinoma (AE1/AE3, RCC, PAX8), rhabdoid tumor (SMA, Desmin), metastatic melanoma (S100, SOX10, HMB-45), and inflammatory myofibroblastic tumor (ALK1) were all negative. A CD68 stain confirmed the large cells to be histiocytes, and special stains for mycobacteria (Ziehl-Neelsen), fungus (GMS), and Michaelis-Gutmann bodies (Von Kossa) were negative. The lymphoplasmacytic infiltrate was composed of κ-restricted CD20-positive B cells and CD138-positive plasma cells. The cytoplasm of macrophages also showed monotypic κ light-chain staining. A clonal IgH rearrangement was detected by multiplex polymerase chain reaction, while MYD88 L265P mutation testing and fluorescence in situ hybridization for MALT1 (18q21) rearrangement were negative. A diagnosis of crystal-storing histiocytosis associated with a low-grade B-cell lymphoma was rendered. A subsequent bone marrow biopsy was negative for involvement by lymphoma, and serum protein electrophoresis showed a slight diffuse increase in gamma globulin without a paraprotein band. With no evidence of other mass lesions by imaging studies, the patient was put on quarterly clinical surveillance and no additional treatment modality was offered. Crystal-storing histiocytosis is a rare entity associated with lymphoproliferative disorders and autoimmune conditions. This case highlights the morphologic and immunohistochemical features of this rare entity (Figure 11).

D. Martínez^1–3 ([email protected]); M. Acosta-Plasencia⁴; Y. He⁴; J. P. Orozco⁵; A. Carrasco¹; A. Altuna-Coy⁴; T. Yang⁴; T. Díaz⁴; T. Molins^2,3,6; R. Ramos^2,3,6; R. M. Marrades^5,7; A. Navarro.^{2–4 1}Department of Pathology, Hospital Clínic, Barcelona; ²Thoracic Oncology Unit, Hospital Clínic, Barcelona; ³Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS); ⁴Molecular Oncology and Embryology Laboratory, Department of Surgery and Medical Specializations, Human Anatomy and Embryology Unit, Faculty of Medicine and Health Sciences, Universitat de Barcelona (UB); ⁵Department of Pneumology, Institut Clínic Respiratori (ICR), Hospital Clínic, Barcelona; ⁶Department of Thoracic Surgery, Hospital Clínic, Barcelona; ⁷Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III, Spain.

Context: Patient-derived lung organoids have gained a significant prominence as valuable tools for studying both cancer and noncancer diseases. However, a consensus on the optimal culture medium has not yet been settled. This study aims to contribute to the ongoing optimization of culture conditions for patient-derived lung organoids.

Design: Tissue samples from tumor and normal paired tissue from 71 patients with early-stage non–small cell lung cancer were used to culture both lung cancer (LCO) and normal lung organoids (LNOs). Organoids were cultured by using the 3 different culture media, which can be classified as Enriched Culture Media (ECM), Limited Culture Media (LCM), or Minimum Culture Media (MCM). Establishment efficiency, viability, and main phenotypic organoid characteristics were analyzed for each medium and compared.

Results: ECM was the most efficient to establish new organoid cell lines from patient samples and supported long-term expansion. In contrast, MCM was less efficient but was unique in that it effectively prevented the growth of LNOs in the LCO cultures. An intermediate result was observed with LCM. Furthermore, the differentiation process occurring to LNOs differed depending on the media used. LNOs cultured in ECM tended to differentiate into bronchospheres, whereas those in LCM also showed alveolosphere differentiation and mesenchymal cell overgrowth. The morphologic characteristics of LCOs were notably influenced by the selected culture media.

Conclusions: This study focused on shedding light on the most suitable culture medium for patient-derived lung organoids, emphasizing the need for tailored conditions to improve organoid model studies. The findings hold significance for lung cancer research and precision medicine applications.

A. V. Teixeira; M. J. F. Do Marco; S. S. Batah; A. J. Rodríguez-Herrera; A. T. Fabro ([email protected]). Department of Pathology and Legal Medicine, Ribeirão Preto, São Paulo, Brazil.

Context: Pleuroparenchymal fibroelastosis (PPFE) is characterized by intra-alveolar fibrosis and elastosis of the alveolar walls, predominantly upper lobe pleural and subjacent parenchymal. While the morphologic patterns and multiple etiologies have been associated with PPFE, our aim was to demonstrate morphologic evidence of the bronchiolar role in the histogenesis of PPFE.

Design: Morphologic analysis of H&E staining and clinical data collection from 12 patients with pneumothorax-related PPFE were included between 2016 and 2024.

Results: All included patients were asymptomatic and experienced spontaneous pneumothorax requiring pleuroparenchymal resection. Most had a history of drug use, smoking, or previous respiratory atopic disease. Pathologic analysis revealed morphologic diagnosis of PPFE. All cases exhibited some degree of bronchiolar remodeling with architectural distortion. The pleura-bronchiolar distance was significantly reduced.

Conclusions: The delineated bronchiolar remodeling adjacent to PPFE may suggest a more active pathogenic role in elastic fiber overproduction. Primary bronchiolar disease may alter tension forces silently or subclinically and lead to pneumothorax in genetically susceptible individuals.

This work was funded by São Paulo Research Foundation (FAPESP) process numbers 23/10184-3, 23/04199-8, 23/10186-6, 22/02821-0, 21/09024-6, 19/01517-3; and National Council for Scientific and Technological Development (CNPq) process number 310415/2021-7.

F. Elsner^1,7 ([email protected]); B. Polzer^1,2; N. Wendler¹; R. Fahrioglu Yamaci¹; C. Unterberger^1,9; B. Cucuruz¹; Z. Czyz¹; G. Feliciello²; M. Hoffmann²; B. Alberter²; S. Treitschke²; P. Rümmele^7,8; W. Sienel³; M. Lindner⁴; H. S. Hofmann⁵; B. Passlick⁶; C. A. Klein.^{1,2 1}Chair for Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany; ²Division of Personalized Tumor Therapy, Fraunhofer ITEM-R, Regensburg, Germany; ³Department of Thoracic Surgery, University of Munich, Grosshadern Campus, Munich, Germany; ⁴Department of Surgery, Asklepios Fachkliniken München-Gauting, Gauting, Germany; ⁵Department of Thoracic Surgery, University of Regensburg, Regensburg, Germany; ⁶Department of Thoracic Surgery, University Medical Center Freiburg, Freiburg, Germany; ⁷Institute of Pathology, University Hospital Erlangen, Erlangen, Germany; ⁸Institute of Pathology, University of Regensburg, Regensburg, Germany; ⁹Fraunhofer Headquarters, Munich, Germany.

Context: The prognostic impact of disseminated cancer cells (DCCs) in non–small cell lung cancer (NSCLC) is still under debate. The aim of this study was (1) to investigate the combined role of DCCs in bone marrow (BM) and lymph node (LN) as prognostic factors in NSCLC and (2) to obtain insights into the biology of metastatic cancer progression.

Design: LN and BM samples of 119 patients with operable NSCLC were screened for DCCs by immunocytology (IC) using antibodies against epithelial cell adhesion molecule (EpCAM) for LN and against cytokeratins 8/18/19 for BM samples. Extraordinary long-term follow-up (median, 59.7 months; range, 1–228 months) was performed until death of all patients. Overall survival (OS) was determined by using Kaplan-Meier and Cox-regression analyses. Comparative genomic hybridization and targeted Sanger sequencing were used for molecular analysis of isolated single DCCs and PTs.

Results: DCCs in LN and/or BM were detected in 41.2% of patients and were associated with significantly reduced OS (P = .004). In subgroup analysis, LN-DCCs were the most important risk factor for reduced OS in pN0 patients and BM-DCCs in pN1-3 patients. Interestingly, PTs and DCCs as well as LN-DCCs and BM-DCCs differed significantly in their genomic alterations, reflecting an ongoing evolutionary process at ectopic sites.

Conclusions: In the future, IC of BM and LN might be a valuable tool to select patients with NSCLC who are at risk and who might benefit from adjuvant therapy. Molecular analysis of DCCs might reveal novel insights into systemic cancer progression and could uncover molecular targets for future therapies.

A. J. Rodríguez-Herrera¹; S. S. Batah¹; M. J. F. do Marco¹; D. T. Wada²; S. B. Silva²; A. T. Fabro¹ ([email protected]). ¹Department of Pathology and Legal Medicine, ²Oncology Division, Department of Medical Images, Hematology and Oncology, Ribeirão Preto Medical School, University of São Paulo, Brazil.

Context: Dendritic cells (DCs) play a pivotal role in an immunoregulatory network, offering DC-based antitumor immunity. While DC vaccine and immunotherapy hold promise for enhancing current clinical benefits in lung cancer, our aim was to assess the diversity of functionally distinct DC subsets in lung cancer and their impact on survival.

Design: Nineteen squamous cell carcinoma (SCC) and 25 adenocarcinoma lobectomy samples at stage 1 were selected between 2007 and 2021. An immunohistochemical panel for CD1a, Langerin, and mannose was conducted, followed by morphometric analysis.

Results: Mannose⁺DCs were present in the invasion front of all non–small cell lung cancers. CD1a⁺DCs were associated with a higher degree of inflammation by histologic analysis. However, both DC subsets were not significantly involved in SCC, and did not affect survival. CD1a⁺DCs and Langerin⁺DCs in adenocarcinomas appeared ineffective in the tumor and front areas. Survival analysis revealed that CD1a⁺DCs exhibited the poorest survival rates in adenocarcinomas with necrotic areas less than 50% (P < .05).

Conclusions: The immune expression of different DC subtypes in non–small cell lung cancers suggests an active tumor microenvironment, favoring tumor progression. Stimulation of certain DC immunophenotypes, such as CD1a⁺ and Langerin⁺, may contribute to promising therapies and may be useful as potential survival biomarkers.

This work was funded by São Paulo Research Foundation (FAPESP) process numbers 23/10184-3, 23/04199-8, 23/10186-6, 22/02821-0, 21/09024-6, 19/01517-3; National Council for Scientific and Technological Development (CNPq) process number 310415/2021-7; and FAEPA 639/2023.

P. Batra ([email protected]); L. Liu. Department of Pathology, University of Chicago (Northshore/Endeavor), Illinois.

Mediastinal mature teratomas are benign germ cell tumors. Rarely, they develop a component of somatic-type malignancy termed germ cell tumors with a somatic-type malignancy. Rhabdomyosarcoma is the most frequent sarcoma. Colonic adenocarcinoma is the most common carcinoma. Neuroendocrine tumors as a somatic-type malignancy are extremely rare. Here, we report a case of mature teratoma with typical carcinoid tumor. A 70-year-old woman presented to the emergency department with chest pain, neck pain, and recurrent atrial fibrillation. During workup, a contrast-enhanced computed tomography of the chest showed a curvilinear, multilobulated, 5.5 x 3.9-cm anterior mediastinal mass. Positron emission tomography revealed mild 18F-fluorodeoxy glucose (FDG) avidity in the mass. She underwent complete mediastinal mass resection. Histopathologic examination showed mature teratoma including areas of cartilage, fibroadipose and smooth muscle tissue, and cysts lined by squamous, respiratory, and intestinal-type epithelium. Nests of low-grade neuroendocrine tumor cells were identified diffusely through the entire mass. The mitotic count was low (<1/2 mm²). No necrosis was seen. The tumor cells were positive for CK8/18, synaptophysin, and chromogranin and negative for PAN-CK, Sall 4, PTH, and GATA 3. A diagnosis of mature teratoma with typical carcinoid tumor was rendered. This case represents a rare example of mediastinal mature teratoma with nests of carcinoid tumor within the tumor. Radiology and gross examination of the tumor did not suggest the presence of a neuroendocrine component, hence thorough sampling of the tumor is necessary to avoid overlooking small lesions and nests of carcinoid tumor (Figure 12, A through D).

H. Shirsat¹ ([email protected]); Shaun Coughlin²; Tony Ng.^{3 1}Department of Pathology, and ²Department of Thoracic Surgery, Royal Jubilee Hospital, Victoria, British Columbia, Canada; ³Department of Pathology, Vancouver General Hospital, Vancouver, British Columbia, Canada.

A 59-year-old woman with secondhand smoke history presented with a large left hilar mass. Biopsy showed a malignant spindle cell proliferation positive for AE1/AE3, CK5/6, p40, and CK7, while negative for CD34 and S100. A diagnosis of sarcomatoid squamous cell carcinoma was rendered. Subsequent pneumonectomy showed a 9.8-cm mass with endobronchial involvement obstructing the lower lobe bronchus and involving mainstem bronchus. Histology demonstrated areas of well-differentiated epithelial myoepithelial carcinoma blending with a high-grade sarcomatous component showing sheets of spindled cells in myxoid stroma with focal osteoid. Myoepithelial cells in the well-differentiated component stained for p63, p40, smooth muscle myosin, and the epithelial component stained for CK7. The sarcomatoid area showed focal positivity for smooth muscle myosin, p40, and p63. Next-generation sequencing detected the pathogenic variants HRAS Q61R and PIK3CA H1047R. Findings confirmed high-grade transformation/dedifferentiation of epithelial myoepithelial carcinoma. Within a year, the patient developed metastases to the liver, spine, and pleura, and died. While epithelial myoepithelial carcinoma has been described in the lung, to our knowledge there has been no report of high-grade transformation in the lung, as may be seen in the head and neck region. Importantly, surgery is curative for salivary gland neoplasms in the lung with only rare instances of distant metastasis. This case highlights that although exceedingly rare, dedifferentiated component of a salivary gland tumor should be considered in the differential diagnosis of spindle cell neoplasms in the hilar region (Figure 13).

S. S. Batah¹; A. J. Rodríguez Herrera¹; M. J. F. do Marco¹; W. M. Telini¹; A. P. Leme²; R. R. Domingues²; R. F. Carvalho³; A. P. Schnepper³; A. A. Cetlin⁴; D. T. Wada⁵; M. K. Santos⁶; T. R. Nadai⁶; J. A. Baddini-Martinez⁷; V. L. Capelozzi⁸; A. T. Fabro¹ ([email protected]). ¹Department of Pathology and Legal Medicine, Ribeirão Preto Medical School, University of São Paulo, Brazil; ²LNBio, Brazilian Center for Research in Energy and Materials (CNPEM), Brazilian Biosciences National Laboratory, Campinas, SP, Brazil; ³Institute of Biosciences, São Paulo State University, Botucatu, Brazil; ⁴Pulmonary Division, Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo, Brazil; ⁵Department of Medical Images, Hematology and Oncology, Ribeirão Preto Medical School, University of São Paulo, Brazil; ⁶Bauru Medical School, University of São Paulo, Brazil; ⁷São Paulo Medical School, Federal University of Sao Paulo, Brazil; ⁸Department of Pathology, Faculty of Medicine, University of São Paulo, Brazil.

Context: The dilation of alveolar sacs with mucoid impaction or Capelozzi necrosis may result from traction caused by bronchiolar remodeling in airway-centered interstitial fibrosis. While the role of pneumocytes in the fibrogenic process is critical, our aim was to explore the protein expression profile in pneumocytes adjacent to mucoid impaction and Capelozzi necrosis.

Design: Twenty-one lung surgical biopsies with alternative diagnosis for usual interstitial pneumonia and a definitive diagnosis of fibrosing interstitial lung diseases (f-ILDs) (hypersensitivity pneumonitis, microaspiration-related interstitial fibrosis, and collagen vascular disease), after MDD by American Thoracic Society/European Respiratory Society (ATS/ERS) criteria, were selected from 2017 to 2021. Laser microdissection (Leica) and mass spectrometry (LTQ Orbitrap Velos Thermo Scientific Velos) were performed in all cases.

Results: A total of 925 distinct peptides were identified, with 12 exclusive to mucoid-only cases (7) and 4 exclusive to Capelozzi necrosis-only cases (4). Nine cases exhibited both mucoid impaction and Capelozzi necrosis, with 17 exclusive peptides found in mucoid impaction and 50 in Capelozzi necrosis. The proteomic profile suggests that peptides from Capelozzi necrosis are more closely related to adaptation and repair, while immunity is more associated with mucoid impaction.

Conclusions: While peribronchiolar remodeling with alveolar sac dilation appears morphologically similar regardless of etiology, the pneumocytes adjacent to mucoid impaction and Capelozzi necrosis demonstrated a range in the etiology-related protein expression profile.

This work was funded by São Paulo Research Foundation (FAPESP) process numbers 23/10184-3, 23/04199-8, 23/10186-6, 22/02821-0, 19/01517-3; and National Council for Scientific and Technological Development (CNPq) process number 310415/2021-7.

A. Zhang¹; A. Galbraith²; V. Akbari²; W. Lam³; D. Huntsman^1,4; S. Jones²; S. Yip^1,5; J. Naso^1,5 ([email protected]). ¹Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada; ²Canada’s Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, British Columbia, Canada; ³British Columbia Cancer Research Institute, Vancouver, British Columbia, Canada; ⁴Department of Molecular Oncology, University of British Columbia, Vancouver, British Columbia, Canada; ⁵Department of Pathology and Laboratory Medicine, Vancouver General Hospital, Vancouver, British Columbia, Canada.

Context: The lungs are one of the most common sites of metastasis. Differentiating primary lung tumors from metastases can affect clinical management. This is especially challenging for squamous cell carcinomas (SqCCs), which are common primary lung tumors but can also metastasize from other sites with overlapping histologic and immunohistochemical features. Epigenetic signatures, including DNA methylation, differ between cells from different tissue lineages and sites. We hypothesized that methylation profiling could distinguish between primary and metastatic SqCCs.

Design: We trained a neural network to predict primary site from array-based DNA methylation data for 1851 primary and metastatic SqCCs (head/neck, pulmonary, cervical, and esophageal origins), and urothelial carcinomas, given their morphologic overlap. Accuracy was assessed on a held-out validation set (n = 474), normal tissue (n = 118), and Nanopore sequencing data for 6 metastases.

Results: Unsupervised clustering revealed 5 major clusters of carcinomas associated with primary site and HPV infection. Our network correctly classified 467/474 carcinomas (99%; 92/92 from lung), including 30/31 metastases to the lung, and was robust to batch effects. The same network correctly classified 113/118 (96%; 42/42 from lung) normal samples, showing recognition of site-intrinsic patterns. The most informative genes used by the classifier were involved in anatomic patterning and lineage specification. The classifier was directly applicable to low-depth Nanopore sequencing data, concordant with consensus-determined primary site in 5/6 metastatic SqCCs.

Conclusions: Leveraging site-intrinsic methylation patterns, our classifier accurately determines the site-of-origin in SqCC. Our approach shows transferability to low-depth Nanopore sequencing data, which could enable rapid, cost-effective primary site determination and inform optimal patient management.

Yip is a member of advisory boards of Amgen, AstraZeneca, Bayer, Janssen, Pfizer, and Roche.

C. Y. Chow¹ ([email protected]); S. Kesavan¹; B. H. Ong²; A. Takano.^{1 1}Department of Anatomical Pathology, Singapore General Hospital; ²Department of Cardiothoracic Surgery, National Heart Centre Singapore.

A 39-year-old nonsmoking woman was found to have a posterior mediastinal mass during routine health screening. Radiologic evaluation revealed a heterogeneously enhancing pleural-based tumor. Surgical intervention revealed a 3-cm posterior mediastinal mass abutting the right upper and lower lobes of the lung. Gross examination showed an encapsulated, cystic tumor with a pushing border into the lung parenchyma. Histologic study demonstrated a biphasic tumor comprising bland spindle cells with myxoid stroma, punctuated by glandular and cribriform epithelial structures. Immunohistochemical stains were strongly positive for S100 and GLI1 in the spindle cells, while being negative for SOX10. The epithelial component was positive for EMA and pan-cytokeratins, as well as TTF-1 nuclear expression. The Ki-67 proliferation index was low, approximately 5% to 10%. Strong GLI1 immunoexpression prompted molecular analysis using next-generation sequencing–based anchored multiplex PCR (Archer FusionPlex), which revealed ACTB::GLI1 gene fusion. Based on these findings, a diagnosis of atypical epitheliomesenchymal biphasic neoplasm with GLI1 fusion was rendered. While the tumor’s biological behavior remains uncertain, the risk of metastasis and local recurrence necessitates close clinical monitoring, akin to that for sarcoma management. This case study highlights that the clinicopathologic spectrum of GLI1-altered tumors is broader than what was originally described in a subset of pericytomas. Identifying a GLI1 alteration in this unique tumor entity may potentially lead to new therapeutic strategies, as tumors with GLI1 oncogenic activation and subsequent PTCH1 overexpression might be sensitive to Sonic Hedgehog (SHH) pathway inhibitors.