TTF-1 Expression Research Articles

Abstract PR005: Leveraging mass cytometry for phenotyping CTCs in SCLC liquid biopsies: Tracking therapy resistance at a personalized level

Abstract Liquid biopsies present a promising, minimally invasive procedure through which clinicians can assess and monitor disease status by interrogating circulating tumor cell (CTC) and immune cell phenotypes. In the case of Small-cell lung cancer (SCLC) – a type of lung cancer characterized by aggressive growth, dismal prognosis and limited treatment options - leveraging liquid biopsies and CTCs for evaluating disease status would be of great benefit, given that biopsies are scarce. Furthermore, SCLC CTC numbers are higher compared to other cancers, presenting a unique opportunity to utilize them for assessing SCLC therapy resistance and identifying new targetable markers. In this study, we leverage mass cytometry (i.e. CyTOF) to phenotype SCLC CTC phenotypes in longitudinal clinical specimens to assess therapy resistance at a personalized level. By using CyTOF - a multiplex cytometric approach that enables interrogation of 30-50 protein markers per single cell – we show that we are able to identify and analyze a significant number of CTCs and their heterogeneous expression profiles. To achieve this, we first optimized a CyTOF panel of ∼25-30 antibodies by using SCLC cell lines and blood specimens from healthy donors and SCLC patients. We show that by using this approach, we are able to identify and phenotype SCLC CTCs by using the combination of CD45 (negative expression) and CD56 positive expression with further validation achieved by TTF1 and SCLC subtype transcription factor expression (NEUROD1, POU2F3 and ASCL1). This approach avoids enriching for specific CTC epithelial phenotypes that express surface markers that fluctuate during epithelial- mesenchymal transition (EMT) (e.g. EpCAM, a common marker for isolating CTCs). For parallel immune profiling we assessed among others CD8/CD4 (T cells) and CD11b/HLA-DR (Myeloid-derived suppressor cells, MDSCs) expression. We then proceeded to interrogate CTC phenotypic differences in the naïve vs relapsed setting as well as dynamic changes in longitudinal blood specimens from SCLC patients by evaluating markers that describe immune suppression activity (PD-L1) and EMT status (E-Cadherin, Vimentin, MUC1, Twist and Slug). Importantly, we show that with our approach we can track in real time therapeutic targets that are currently being tested in clinical trials or have been recently FDA approved (e.g. pYAP and DLL3) and found that MUC1 and pYAP are significantly increased in CTCs detected in blood specimens of SCLC relapsed patients. Our immune profiling showed an increase of MDSC percentages and a decrease in the CD4/CD8 T cell ratio in the relapsed setting. Finally, when we used PHENOSTAMP, a previously developed reference EMT map for assessing EMT phenotypes in lung cancer clinical specimens, we observed an increase of EMT heterogeneity in CTCs in relapsed patients. Our study highlights the translational power of our approach in utilizing CyTOF for longitudinally tracking CTCs directly in SCLC patient liquid biopsies towards assessing therapy response and resistance at a personalized level. Citation Format: Loukia G Karacosta, Sayantan Bhattacharyya, Allison Stewart, Ashley Victorian, Ester F Lujan, Shafqat Ehsan, Subin Kim, Alberto Duarte, Runsheng Wang, Benedict Anchang, Jing Wang, Lauren Byers. Leveraging mass cytometry for phenotyping CTCs in SCLC liquid biopsies: Tracking therapy resistance at a personalized level [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR005.

Biological and clinical characteristics of non-small cell lung cancer non-specific subtype

BackgroundNon-small cell lung cancer-not otherwise specified (NSCLC-NOS) is a rare subtype of NSCLC that cannot be classified specifically based on morphology and/or special staining. This study aimed to explore the clinical features, biological and pathological characteristics, treatment, and prognosis of NSCLC-NOS. MethodsThis retrospective study included NSCLC-NOS patients diagnosed and treated between 2010 and 2022. Clinical features, gene expression, first-line treatment, and prognosis were analyzed. Kaplan-Meier methods were calculated and log-rank tests and univariable and multivariable Cox regression analyses were performed to determine the relationship between prognostic factors and survival. ResultsOf 105 NSCLC-NOS patients, most were male (92.4%), smokers (78.1%), with a median age of 64 years, and advanced stage (IIIc-IV, 72.4%). Immunohistochemical analysis showed minimal expression of p40, NapsinA, and TTF-1, whereas cytokeratin (CK) was expressed in 100% of cases. 20.5% of 39 patients who underwent genetic testing had driver gene mutations, including EGFR, KRAS, and ROS1. Among 69 patients with complete treatment information, 58 received platinum-based chemotherapy, with paclitaxel being the most commonly used combination chemotherapy drug (n=25), followed by pemetrexed (n=21). The objective response rate (ORR) of paclitaxel was found to be higher compared to pemetrexed (83.3% vs. 54.5%, P=0.296). Furthermore, the combination of paclitaxel with immunotherapy demonstrated superior benefits in comparison to pemetrexed (76.9% vs. 50.0%, P=0.367). The median progression-free survival (PFS) for patients treated with monotherapy paclitaxel, the paclitaxel-immunotherapy combination, and the pemetrexed-immunotherapy combination were 6.6 months (95% CI: 1.508-11.692; P=0.017), 15.7 months (95% CI: 14.071-17.329; P=0.017), and 11.8 months (95% CI: 10.279-13.321; P=0.324), respectively. The median overall survival (OS) was 13.6 months. Anatomic location (P=0.026) and immunotherapy use (P=0.003) were associated with OS. Multivariate analysis confirmed that anatomical location and immunotherapy use were factors influencing the prognosis. ConclusionNSCLC-NOS is common in male smokers and often diagnosed at an advanced stage with low mutation rate. Paclitaxel with immunotherapy may have better benefits as a first-line treatment. Anatomic location and immunotherapy use are prognostic factors.

Exploring the clinicopathological characteristics of submucosal tumor-like esophageal squamous cell carcinoma and the diagnostic significance of endoscopic ultrasound: a comprehensive analysis.

Completely intramural growth submucosal squamous cell carcinoma of the esophagus, also known as SMT-like esophageal squamous cell carcinoma (ESCC), represents a rare and distinct form of esophageal cancer. Its white light endoscopic manifestations resemble those of esophageal subepithelial lesions, and biopsy pathology is often negative, leading to potential oversight or misdiagnosis. This study aimed to comprehensively summarize the clinicopathological and endoscopic ultrasound (EUS) characteristics of patients with SMT-like ESCC while also evaluating the immunohistochemical expression of these patient. This study collected clinical data, including demographic and clinicopathological data, as well as EUS findings, from six patients with SMT-like ESCC. Immunohistochemical analysis was also conducted on tumor tissues to assess the expression of CK7, CK19, CK20, TTF-1, SMA, S-100, Melan-A, CD117, Mucin (MUC) 2, and MUC5. In EUS, SMT-like ESCC is characterized by nonuniform hypoechoic lesions with indistinct borders, often exhibiting a burr or serrated appearance. Most of these lesions involved multiple levels. Cytological specimens obtained through EUS-guided fine needle aspiration (EUS-FNA) revealed suspected squamous cell carcinoma with positive expression of CK5/6, P40, and P63, further confirming the diagnosis of ESCC. Additionally, four patients exhibited CK7+/CK20- immune-expression profiles, and all patients had positive CK19 expression. TTF-1, SMA, S-100, Melan-A, CD117, MUC2, and MUC5 were negative. Combining EUS with EUS-FNA is a valuable approach for diagnosing and differentiating SMT-like ESCC. Furthermore, the characteristic CK7+/CK20- immune profile suggested a potential origin from the esophageal submucosa glands.

Expression of p63 and TTF1 in Non-small Cell Lung Carcinoma with Special Reference to Mutation of EGFR and ALK in Adenocarcinoma of the Lung: An Institutional Experience

Expression of p63 and TTF1 in Non-small Cell Lung Carcinoma with Special Reference to Mutation of EGFR and ALK in Adenocarcinoma of the Lung: An Institutional Experience

Relationship between the expressions of DLL3, ASC1, TTF-1 and Ki-67: First steps of precision medicine at SCLC.

This study presents an observational, cross-sectional analysis of 64 patients diagnosed with small cell lung cancer (SCLC) at a reference laboratory for thoracic pathology between 2022 and 2024. The primary objective was to evaluate the expression of Delta-like ligand 3 (DLL3) and other neuroendocrine markers such as Chromogranin, and Synaptophysin, utilizing both traditional immunohistochemistry and digital pathology tools. Patients were primarily older adults, with a median age of over 71, and most biopsies were obtained from lung parenchyma. Immunohistochemistry (IHC) was performed using specific monoclonal antibodies, with DLL3 showing variable expression across the samples. Notably, DLL3 was expressed in 72.3% of the cases, with varied intensities and a semi-quantitative H-score applied for more nuanced analysis. ASCL1 was expressed in 97% of cases, with the majority considered low-expressors. Only 11% had high expression. TTF-1, traditionally not a conventional marker for the diagnosis of SCLC, was positive in half of the cases, suggesting its potential as a biomarker. The study underscores the significant variability in the expression of neuroendocrine markers in SCLC, with implications for both diagnosis and potential therapeutic targeting. DLL3, particularly, shows promise as a therapeutic target due to its high expression rate in the cohort. The use of digital pathology software QuPath enhanced the accuracy and depth of analysis, allowing for detailed morphometric analysis and potentially informing more personalized treatment approaches. The findings emphasize the need for further research into the role of these markers in the management and treatment of SCLC, considering the poor prognosis and high mortality rate observed in the cohort.

8637 Pituicytoma mimicking Craniopharyngioma

Abstract Disclosure: O. Araoye: None. T. Delibasi: None. A. Chaugule: None. S.K. Dhillon: None. C. Fuller: None. Pituicytoma, a rare low-grade glial tumor originating from the neurohypophysis or infundibulum of the pituitary gland, poses diagnostic challenges due to its propensity to mimic other sellar or suprasellar lesions. We present a case of a 51-year-old male with a history of a suprasellar mass initially suggested to be a craniopharyngioma, incidentally discovered. Opting for elective surgery, the patient underwent transsphenoidal resection, revealing a highly vascular tumor adherent to the optic chiasm and pituitary stalk. While the tumor was successfully removed, sacrifice of the pituitary stalk led to postoperative complications, including cerebrospinal fluid (CSF) leak and central diabetes insipidus (DI).Histopathological examination characterized the tumor as oncocytic pituicytoma/spindle cell oncocytoma, a subtype of pituicytoma. The anti-mitochondrial antibody staining at an external laboratory displayed diffuse granular cytoplasmic staining supporting oncocystic pituicytoma/spindle cell oncocytoma.Postoperatively, the patient developed central DI treated initially with desmopressin (DDAVP), complicated by subsequent hyponatremia necessitating salt tablets and fluid restriction. Additionally, central adrenal insufficiency was managed with hydrocortisone, and central hypothyroidism with levothyroxine. A history of testosterone deficiency was addressed through testosterone injections every 10 weeks.Pituicytoma's diagnostic complexity arises from nonspecific radiological features, requiring reliance on histology and immunohistochemistry for definitive diagnosis. The expression of TTF1 emerges as a characteristic marker. Surgical resection, the primary treatment modality, proves curative in most cases, albeit with associated morbidity including CSF leak, DI, and panhypopituitarism. The role of adjuvant radiotherapy remains uncertain, potentially considered in cases of incomplete resection or recurrence.This case emphasizes three key aspects: first, the potential misdiagnosis of a lobulated suprasellar mass as craniopharyngioma when it could be pituicytoma; second, the development of postoperative DI due to pituitary stalk involvement; and third, the importance of considering pituicytoma in the differential diagnosis of sellar and suprasellar masses.In conclusion, pituicytoma, though rare, demands a nuanced diagnostic approach and comprehensive postoperative management to address potential complications and optimize patient outcomes. Presentation: 6/2/2024

Yi-Fei-San-Jie Chinese medicine formula reverses immune escape by regulating deoxycholic acid metabolism to inhibit TGR5/STAT3/PD-L1 axis in lung cancer

BackgroundYi-Fei-San-Jie Formula (YFSJF), a proprietary medicine of the First Affiliated Hospital of Guangzhou University of Chinese Medicine, has been widely used in clinical practice for several years and is currently being tested in randomized controlled trials for early-stage lung cancer in China. However, the mechanisms by which YFSJF affects lung cancer biology, particularly the immune microenvironment and metabolic processes, remain poorly understood. PurposeThis study aims to explore how YFSJF modulates the immune microenvironment and metabolism in lung cancer, specifically its unique role in inhibiting immune evasion by targeting the TGR5/STAT3/PD-L1 pathway, which has not previously been reported. MethodsComputed Tomography (CT) scan was used to assess YFSJF efficacy in patients with lung cancer and a mouse model of urethane-induced lung cancer. Histopathological evaluation, flow cytometry, and metabolomic analysis were used to assess lung tissue structure, immune cell subset changes, and metabolism modulation, respectively. Western blotting and immunohistochemistry were used to detect Ki67, TTF-1, TGR5, STAT3, p-STAT3, and PD-L1 protein expression. Serum cytokines were detected by ELISA. ResultsYFSJF effectively reduced the size of human lung cancer lesions and decreased the tumor burden and improved survival rates in mice. Lung tissue structure was also improved after YFSJF treatment. YFSJF regulated T-cell subsets, particularly by downregulating cells with PD-1-positive expression of CD3+, CD4+, and CD8+, and elevated serum TNF-α, IFN-γ, and GzmB levels. In addition, YFSJF modulated bile acid metabolism, particularly by inhibiting deoxycholic acid metabolism, which participates in immune regulation in lung cancer by acting on the G protein-coupled bile acid receptor TGR5. ConclusionFinally, YFSJF inhibited immune evasion by blocking the TGR5-mediated STAT3/PD-L1 pathway, weakening PD-L1 and PD-1 binding and reviving T-cell immune activity, thereby countering lung cancer immune evasion and exerting anti-tumor effects.

Clinicopathologic Analysis of HPV-Related Primary Squamous Cell Carcinoma of the Thyroid.

This study aims to explore the clinical and pathologic characteristics of HPV-related primary thyroid squamous cell carcinoma (PSCCT), a rare tumor classified by WHO-5 as a subtype of anaplastic thyroid carcinoma (ATC). Clinical data, histomorphology, immunohistochemistry, HPV detection, and B-raf gene point mutations of 3 PSCCT cases were analyzed. Subsequent follow-up was conducted post-treatment. All 3 cases involved female patients aged between 60 and 76. Microscopic examination revealed squamous cell carcinoma in cases 1 and 3, whereas case 2 exhibited both squamous cell carcinoma and papillary thyroid carcinoma components. Immunohistochemistry demonstrated CK19, PAX8, and TTF1 expression in the papillary thyroid carcinoma component, and CK5/6, p63, p40, and PAX8 expression in the squamous cell carcinoma component. P16 exhibited diffuse positivity in both squamous cell carcinoma and classic papillary carcinoma. HPV analysis identified low-risk type 6 positivity in cases 1 and 3, while both squamous cell carcinoma and papillary carcinoma areas in case 2 were positive for HPV-33. B-raf gene mutation was exclusive to case 2. Diagnosis of PSCCT necessitates multidisciplinary assessment, incorporating clinical symptoms, imaging, histomorphology, and immunohistochemistry. This study, for the first time, reveals the presence of HPV DNA in both PTC and PSCCT, occurring concurrently but separately. Given the limited scope of 3 case reports, definitive conclusions cannot be drawn, warranting further investigation.

Clinical characteristics of KRAS mutation subtypes in non-small cell lung cancer population in Xinjiang, China, and their impact on the prognosis of immunotherapy

PurposeNon-small cell lung cancer (NSCLC) is a highly fatal malignancy. The Kirsten rat sarcoma viral oncogene (KRAS) gene profoundly impacts patient prognosis. This study aims to explore the correlation between KRAS mutation subtypes, clinical data, and the impact of these subtypes on immunotherapy.Materials and methodsTumor samples from 269 NSCLC patients at the Affiliated Cancer Hospital of Xinjiang Medical University were analyzed. Patients received first- or second-line therapy without targeted therapy. Molecular and clinical data were used to analysis KRAS mutation subtypes and treatment outcomes.ResultsKRAS mutations predominantly included G12C, G12D, and G12V subtypes. TP53 had the highest mutation frequency among KRAS mutations, followed by MST1, STK11, and KMT2C. Gender differences were noted among KRAS mutation subtypes, with G12C and G12V mutations prevalent in males, while G12D mutations were less common among males. Smokers exhibited varied KRAS mutation subtypes, with G12C and G12V prevalent in smokers and G12D in nonsmokers. KRAS mutations were mainly in lung adenocarcinoma. TTF-1 and PD-L1 expression differed significantly among KRAS mutations. Patients with G12C and G12V mutations showed higher TMB levels and better immunotherapy outcomes compared to those without KRAS mutations. Conversely, patients with G12D mutations had poorer immunotherapy responses.ConclusionsKRAS mutation subtypes exhibit distinct clinical and molecular characteristics and varying responses to immunotherapy. G12C and G12V mutations correlate with better immunotherapy outcomes, while G12D mutations are associated with poorer responses.

Clinical significances of TTF-1, neuroendocrine (chromogranin, synaptophysin, CD56), and keratin (pancytokeratin, CK7, CK5/6) marker immunostaining in small cell lung cancer.

Immunohistochemistry (IHC) markers have established a role in the pathological diagnosis of small cell lung cancer (SCLC) and especially neuroendocrine markers help to differentiate SCLC from other tumors. The study aimed to evaluate the clinical role of different IHC markers in SCLC patients. A total of 378 SCLC patients were enrolled in the study and analyzed retrospectively. TTF-1, neuroendocrine markers (chromogranin, synaptophysin, and CD56), and keratin markers (pancytokeratin, CK7 and CK5/6) were assessed. CD56 had the highest expression (92.3%) followed by pancytokeratin (82.8%), TTF-1 (74.8%), synaptophysin (72.7%), chromogranin (55.6%), CK7 (54.8%), and CK5/6 (9%). No differences were observed in the expression of all markers according to the stage of the disease. Extended disease SCLC (ED-SCLC) patients with synaptophysin expression had a higher response to chemotherapy compared to those without staining (p = 0.01); on the other hand, the chemotherapy response of these patients was not significantly different when they expressed CK7 (p = 0.06). Pancytokeratin expression was associated with favorable survival in both limited disease SCLC (LD-SCLC) (p = 0.02) and ED-SCLC (p = 0.005) patients. Similarly, ED-SCLC patients with CD56 staining lived longer than those without expression (p = 0.001). The lack of synaptophysin expression in LD-SCLC patients (p = 0.06) and TTF-1 expression in ED-SCLC patients (p = 0.06) were correlated with better survival rates. We conclude that IHC markers, used frequently in the diagnosis of SCLC, might also be used in clinical decision-making, since they are correlated with predictive and prognostic factors for the disease.

Isolated Monoclonal T-Cell Receptor Gene Rearrangement in a Lung Adenocarcinoma Harboring MET Exon 14 Skipping: Diagnostic Pitfall.

In the diagnostic workup of poorly differentiated tumors, T-cell receptor (TCR) clonality has long been considered as evidence of T-cell lymphoma. MET exon 14 skipping (METex14) is a mutation typically seen in lung adenocarcinoma. Herein, we present the first report of METex14 lung adenocarcinoma with isolated monoclonal TCRγ gene rearrangement. A 69-year-old woman presented to an outside hospital with pleural effusions. A pleural decortication demonstrated malignant cells positive for CD30 and CD138 but negative for BerEP4, KRT5, and EMA. An equivocal HHV8 staining was interpreted as positive, leading to the erroneous outside diagnosis of primary effusion lymphoma. Additional workup at our institution revealed a lack of HHV8 and T-cell markers but the presence of TCRγ clonality, pankeratin, and TTF1 expression. Repeat TCRγ testing on the in-house biopsy was negative for clonality. Next-generation sequencing detected METex14, confirming the diagnosis of lung adenocarcinoma. The potential diagnostic pitfall and prognostic/predictive implications are discussed.

Integrated On-Slide Positive Controls for Immunocytochemistry on Cytology Slides.

Integrated on-slide positive controls are a standard quality assurance and quality control measure for immunohistochemistry on formalin-fixed paraffin-embedded tissue sections. They ensure identical analytical conditions for the control and patient samples. Our aim was to develop a procedure for preparing integrated on-slide positive controls for immunocytochemistry (ICC) on methanol-fixed cytospins. Leftover diagnostic cytology samples with sufficient cells and confirmed expression of Calretinin, MOC31, TTF1, and hormone receptors were used as control samples. Cells from the control samples were deposited on the peripheral part of objective slides using standard cytocentrifuge equipment. Cytospins were immediately fixed in methanol for at least 30 min and then covered with polyethylene glycol (PEG). Completely dry and solid PEG was removed from the central part of the objective slides and stored at room temperature. Patient samples were subsequently added to the central part of a PEG-protected slide, with an appropriate positive control placed on the peripheral part, and then fixed in methanol. ICC was performed on the Ventana/Roche automated platform ULTRA, using optimized and validated protocols for TTF1, hormone receptors, and double immunostaining for Calretinin/MOC31. The quality of ICC reactions for both deposits on the same slide and potential cell carryover was evaluated retrospectively. In the period from October 2021 to December 2023, the majority of integrated positive controls (364/368, 99%) consistently exhibited unequivocally positive reactions for TTF-1 (n = 93), hormone receptors (n = 84), and double staining for Calretinin/MOC31 (n = 191), with easily interpretable ICC reactions on corresponding patient samples. No obvious carryover of cells from the control sample to the patient sample was observed during this period. A novel approach developed for preparing integrated on-slide positive controls for ICC on methanol-fixed cytospins using standard cytocentrifugation is low-cost and can be widely applied in diagnostic cytology laboratories. Simultaneous ICC procedures for the control and patient samples on the same slide ensure identical analytical conditions for both samples, providing the highest level of quality control while reducing costs. Interpreting both ICC reactions on the same slide is time-efficient and convenient.

The utility of the lineage specific immunohistochemical stains SATB2, CDX2, and villin, and the mucin glycoproteins MUC2, MUC5AC, and MUC6 to distinguish pulmonary invasive mucinous adenocarcinoma from metastatic colorectal carcinoma

ContextThe lungs are a common site of tumor metastasis. While morphology and immunophenotype can help differentiate primary from metastatic tumors, distinguishing pulmonary invasive mucinous adenocarcinoma (PIMA) from metastatic colorectal adenocarcinoma (CRC) may occasionally be challenging due to overlapping morphological and immunohistochemical features. Lineage-specific markers such as CDX2, TTF-1, and napsin A are helpful with pulmonary non-mucinous adenocarcinoma (PNMA), however they are non-specific and insensitive when applied to PIMA. SATB2 is a newer marker that distinguishes CRC from upper gastrointestinal and pancreaticobiliary tumors; its utility in distinguishing CRC from PIMA has not been fully elucidated. ObjectiveTo evaluate the performance of lineage-specific and mucin glycoprotein immunostains in distinguishing PIMA and CRC. DesignWe stained tissue microarrays comprising 34 PNMA, 31 PIMA, and 32 CRC with CK7, CK20, SATB2, CDX2, villin, TTF-1, napsin A, and gel-forming mucins MUC2, MUC5AC, and MUC6. ResultsPIMA showed significant (>50% of cells) expression of SATB2 (6%), CDX2 (6%), villin (74%), TTF-1 (13%), and napsin A (23%). However, significant CK7 expression was seen in nearly all PIMA (30/31) and none of the metastatic CRC. ConclusionOur results suggest that CK7 remains one of the most useful markers for distinguishing primary PIMA from metastatic CRC. Expression of the mucin glycoproteins MUC5AC and MUC6 and lack of expression of MUC2 favored a diagnosis of PIMA, but expression of these markers was too heterogeneous to be of clinical utility. To our knowledge this is the only study comparing the immunohistochemical profile of PIMA and metastatic CRC in lung metastasectomy specimens.

Single-Cell Transcriptome Analysis Reveals 2 Subtypes of Tumor Cells of Sclerosing Pneumocytoma With Distinct Molecular Features and Clinical Implications

Pulmonary sclerosing pneumocytoma (PSP) is a rare, distinctive benign lung adenoma of pneumocyte origin. Despite its rarity, the tumor’s unique cellular morphology has sparked ongoing debates regarding the origin of its constituent cells. This study aimed to elucidate the molecular features of PSP tumor cells and enhance our understanding of the cellular processes contributing to PSP formation and biological behavior. Tissue samples from PSP and corresponding normal lung tissues (n = 4) were collected. We employed single-cell RNA sequencing and microarray-based spatial transcriptomic analyses to identify cell types and investigate their transcriptomes, with a focus on transcription factors, enriched gene expression, and single-cell trajectory evaluations. Our analysis identified 2 types of tumor cells: mesenchymal-epithelial dual-phenotype (MEDP) cells and a distinct subpopulation of type II alveolar epithelial cells exhibiting characteristics slightly reminiscent of type I alveolar epithelial cells (AT2Cs) corresponding to histologic round stromal cells and surface cuboidal cells, respectively. MEDP cells displayed weak alveolar epithelial differentiation but strong collagen production capabilities, as indicated by the expression of both TTF-1 and vimentin. These cells played a pivotal role in forming the solid and sclerotic areas of PSP. Moreover, MEDP cells exhibited a pronounced propensity for epithelial-mesenchymal transition, suggesting a greater potential for metastasis compared with AT2Cs. The capillary endothelial cells of PSP displayed notable diversity. Overall, this study provides, for the first time, a comprehensive mapping of the single-cell transcriptome profile of PSP. Our findings delineate 2 distinct subtypes of tumor cells, MEDP cells and AT2Cs, each with its own biological characteristics and spatial distribution. A deeper understanding of these cell types promises insights into the histology and biological behaviors of this rare tumor.

Expression patterns of HNF4α, TTF-1, and SMARCA4 in lung adenocarcinomas: impacts on clinicopathological and genetic features.

HNF4α expression and SMARCA4 loss were thought to be features of non-terminal respiratory unit (TRU)-type lung adenocarcinomas, but their relationships remained unclear. HNF4α-positive cases among 241 lung adenocarcinomas were stratified based on TTF-1 and SMARCA4 expressions, histological subtypes, and driver mutations. Immunohistochemical analysis was performed using xenograft tumors of lung adenocarcinoma cell lines with high HNF4A expression. HNF4α-positive adenocarcinomas(n = 33) were divided into two groups: the variant group(15 mucinous, 2 enteric, and 1 colloid), where SMARCA4 was retained in all cases, and the conventional non-mucinous group(6 papillary, 5 solid, and 4 acinar), where SMARCA4 was lost in 3/15 cases(20%). All variant cases were negative for TTF-1 and showed wild-type EGFR and frequent KRAS mutations(10/18, 56%). The non-mucinous group was further divided into two groups: TRU-type(n = 7), which was positive for TTF-1 and showed predominantly papillary histology(6/7, 86%) and EGFR mutations(3/7, 43%), and non-TRU-type(n = 8), which was negative for TTF-1, showed frequent loss of SMARCA4(2/8, 25%) and predominantly solid histology(4/8, 50%), and never harbored EGFR mutations. Survival analysis of 230 cases based on histological grading and HNF4α expression revealed that HNF4α-positive poorly differentiated (grade 3) adenocarcinoma showed the worst prognosis. Among 39 cell lines, A549 showed the highest level of HNF4A, immunohistochemically HNF4α expression positive and SMARCA4 lost, and exhibited non-mucinous, high-grade morphology in xenograft tumors. HNF4α-positive non-mucinous adenocarcinomas included TRU-type and non-TRU-type cases; the latter tended to exhibit the high-grade phenotype with frequent loss of SMARCA4, and A549 was a representative cell line.

Abstract 6799: The impact of targeting TRAF2 and NCK-interacting protein kinase on anti-tumor effect and tumor immune environment in cMYC-high small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is a highly lethal malignancy with rapidly acquired chemotherapy resistance. While the inflamed SCLC subtype is associated with a significantly improved response to immunotherapy, the non-inflamed SCLC subtypes, encompassing most SCLC patients, experience only modest survival gains with chemo-immunotherapy. Wnt signaling pathway activation is associated with resistance to chemotherapy and an immune checkpoint inhibitor in several tumors. TRAF2 and NCK-interacting protein kinase (TNIK), which interacts with downstream effectors, TCF4/β-catenin transcriptional complex, is an essential activator of Wnt target genes. TNIK is highly expressed in several cancers, but the efficacy of TNIK inhibition in SCLC has not previously been researched. We previously demonstrated that single agent TNIK inhibitors are effective in a subset of SCLC. Here, we hypothesize that targeting TNIK will show an anti-tumor effect based on specific biomarkers and also modify the tumor immune environment in SCLC. Methods: We evaluated susceptibility to a small molecule TNIK inhibitor, NCB-0846, in 29 human-derived SCLC cell lines using 96-hour proliferation assays. To investigate efficacy biomarkers, we correlated NCB-0846 IC50 values with proteomic expression (profiled using reverse phase protein array). cMYC and FOXK1 expression was reduced by siRNA. Chemokine concentrations were determined by ELISA. Results: SCLC cell lines with high cMYC levels, were more sensitive to the TNIK inhibitor, NCB-0846 (F.C.=-2.07, p=0.01), while SCLC cell lines with high expression of TTF-1 were more resistant (F.C.=2.48, p<0.01). Knockdown of cMYC conferred resistance to NCB-0846 in cMYC-high cells, while NCB-0846 treatment resulted in decreased cMYC expression in a concentration-dependent manner. Additionally, NCB-0846 treatment decreased the immunosuppressive chemokine CCL2 levels in the supernatant of human-derived SCLC cell lines and GEMM-derived cell lines. The expression of FOXK1, a transcription factor of CCL2, was also reduced in cell lines treated with NCB-0846. Conclusions: These findings show that TNIK inhibition is more effective in cMYC-high SCLC, acting through downregulating cMYC levels. TNIK inhibition also decreases the production of CCL2, an immunosuppressive chemokine implicated in resistance to anti-PD-L1 immunotherapy. Together, these findings highlight the promising potential of TNIK inhibitors in cMYC-high SCLC. Additionally, suppressing CCL2 by TNIK inhibition supports the rationale for combining the TNIK inhibitor and an anti-PD-L1 antibody in the non-inflamed SCLC subtypes. Citation Format: Azusa Tanimoto, Kavya Ramkumar, Allison Stewart, Bingnan Zhang, Robert Cardnell, Shen Li, Qi Wang, Jing Wang, Carl Gay, Lauren Averett Byers. The impact of targeting TRAF2 and NCK-interacting protein kinase on anti-tumor effect and tumor immune environment in cMYC-high small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6799.

Premature Classification of Early-stage Endometrioid Ovarian Carcinoma With Mesonephric-like Differentiation as Mesonephric-like Adenocarcinoma.

Ovarian mesonephric-like adenocarcinoma (MLA) is a rare tumor with potential origins in endometriosis and Müllerian-type epithelial tumors. The morphologic patterns of MLA overlap with those of endometrioid ovarian carcinoma (EnOC). We speculated that a subset of MLAs would be classified as EnOCs. In this study, we attempted to identify MLAs from malignant endometrioid tumors. Given that the study patients with MLAs had both endometrioid-like and mesonephric-like morphologies, we defined mesonephric-like differentiation (MLD) as an endometrioid tumor with focal or diffuse MLA morphology and immunophenotype. Twelve patients exhibited mesonephric-like morphologic patterns. Immunohistochemistry analysis for CD10, TTF-1, estrogen receptor (ER), GATA3, calretinin, and PAX8 expression was done using whole-section slides. Two patients without the MLA immunophenotype were excluded. Ten patients with EnOCs with MLD (8.3%) were identified from a cohort of 121 patients with malignant endometrioid tumors. All 10 patients were positive for TTF-1 and/or GATA3. Most patients were ER-negative. Morphologically, MLD was associated with papillary thyroid carcinoma-like nuclei, flattened cells, tubular, nested, reticular, or glomeruloid architecture, and infiltrative growth. All 10 patients had pre-existing endometriosis and/or adenofibromas. Among the EnOCs with MLD, 5 had coexisting components such as EnOC grade 1 [(G1), cases 4, 7, and 9], mucinous borderline tumor (case 1), and dedifferentiated carcinoma (case 10), with distinct borders between EnOC with MLD and the other components. Nine of the 10 MLA patients (90%) harbored KRAS hotspot mutations. In addition, 4 patients harboring other components shared common KRAS hotspot mutations. No significant prognostic differences were observed between patients with and without MLD. Based on our findings, we suggest that EnOC with MLD, especially in the early stages and without high-grade components, should be considered a subtype of EnOC. Overtreatment should be avoided in such patients, particularly in the early stages. In this study, as the characteristics between EnOC with MLD and MLA were not distinguishable, we considered both conditions to be on the same spectrum. EnOCs with MLD exhibit the MLA phenotype during disease progression and are prematurely classified as MLA. Nevertheless, more patients with EnOC who have MLD/MLA are required for a more robust comparison between conventional EnOC according to staging and grading.

Pulmonary gangliocytic paraganglioma: An under-recognized mimic of carcinoid tumor

Gangliocytic paragangliomas are rare neoplasms occurring almost exclusively in the ampullary region of the gastrointestinal tract. Although these tumors are not typically considered in the differential diagnosis of primary pulmonary neoplasia, 5 cases of primary pulmonary gangliocytic paragangliomas have been previously reported. Herein we report our experience with 3 additional examples, all referred to our Anatomic Pathology Consultation service. The patients (a 32-year-old man, a 69-year-old woman and a 55-year-old man) each presented with an endobronchial (2 cases) or upper lobe lung mass, ranging from 1.5 to 2.5 cm in maximum dimension. Biopsy and endobronchial debulking specimens demonstrated the classic triphasic morphology of gangliocytic paraganglioma, with epithelial, spindled and ganglion-like cells. By immunohistochemistry, the tumors were positive for keratin, synaptophysin and chromogranin A in the epithelial component, S100 protein and glial fibrillary acidic protein (GFAP) in the Schwannian spindled cells, and synaptophysin in ganglion cells. TTF1 expression was seen in the epithelial components of 2 cases. The Ki-67 labelling index was low (<2%). Primary pulmonary gangliocytic paragangliomas should be distinguished from carcinoid tumors, given the different natural histories and risk stratification approaches for these morphologically similar tumors. Awareness that gangliocytic paraganglioma may occur in the lung and appropriate immunohistochemical studies are key to correct diagnosis.

18F-FDG PET/CT imaging in pulmonary sarcomatoid carcinoma.

Pulmonary sarcomatoid carcinoma (PSC) is a rare highly aggressive and poorly differentiated non-small cell carcinoma, and little is known about the information on the usefulness of 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT). We investigated the clinical and 18F-FDG PET/CT features of PSC. We retrospectively analyzed 25 consecutive PSC patients who had undergone 18F-FDG PET/CT. Demographic data, PET/CT findings before treatment, pathological features, and prognosis in these patients were investigated to define correlates between maximal standard uptake value (SUVmax) and clinicopathological parameters. From March 2017 to January 2023, twenty-five eligible patients with PSC were identified. There were 23 (92%) men, aged 68.5 ± 8.5 (range 56-90) years. Eighteen (72%) patients had a frequent smoking history. The mean size of PSCs was 59.3 ± 18.6 (range 29-97) mm, and 23 (92%) PSCs were Stage IV tumors. 20 (80%) lesions were located in the upper lung and 19 (76%) cases belonged to the peripheral type. Necrotic foci appeared in 21(84%) tumors. 11 (44%) PSCs invaded the pleura. All PSCs were FDG avid, and the mean of SUVmax was 11.8 ± 5.3 (range 4.8-25.5). Metastases were found on PET/CT in 24(96%) patients. The SUVmax of the lesions ≥ 5cm was higher than that of the lesions < 5cm (p=0.004), and the SUVmax of lesions with TTF-1 expression was higher than those of lesions without TTF-1 expression (p=0.009). All of the 25 primary lesions were considered malignant and confirmative, probable, and possible diagnosis of PSC was made in 2 (8%), 4 (16%), and 5(20%) patients, respectively on PET/CT. PSC was not considered in 14 (56%) patients, in PET/CT. The survival of patients with surgery didn't demonstrate a significantly good prognosis as compared with those without surgery (p=0.675). All PSCs had obvious FDG avidity. Although imaging diagnosis is still difficult, combined clinical and imaging features more than 40% of primary lesions were considered for the possibility of PSC in our group. Early histopathological diagnosis is necessary to help develop a reasonable regimen.

Abstract LB_B05: Mapping therapy resistant phenotypes in SCLC liquid biopsies with single-cell proteomics at a personalized level

Abstract Small-cell lung cancer (SCLC) accounts for ~15% cases of all lung cancer cases and is characterized by aggressive growth and dismal prognosis. Although patients initially respond favorably to platinum and immune checkpoint blockade therapy, most develop therapy resistance and relapse within 1 year. Leveraging liquid biopsies and circulating tumor cells (CTCs) for evaluating SCLC status is regarded as a promising approach especially in SCLC where biopsies are scarce. SCLC CTC numbers are higher compared to other cancers, presenting a unique opportunity to utilize them for assessing SCLC therapy response and resistance and identifying new targetable markers. In this study we leverage mass cytometry (i.e., CyTOF), a multiplex cytometric approach that enables interrogation of 30-50 protein markers per single cell, allowing us to identify and analyze a significant number of CTCs and their heterogeneous expression profiles. Our goal was to utilize CyTOF to help us map changes in CTC expression profiles in the treatment naïve and relapsed setting and thus be able to assess therapy response and resistance at a personalized level. To achieve this, we optimized a CyTOF antibody panel by using SCLC cell lines and blood specimens from healthy donors and SCLC patients. We show that by using this approach, we are able to identify and phenotype SCLC CTCs by using the combination of CD45 (negative expression) and CD56 positive expression with further validation achieved by TTF1 and SCLC subtype transcription factor expression (e.g. NEUROD1, POU2F3). This approach avoids enriching for specific CTC epithelial phenotypes that express surface markers that fluctuate during epithelial-mesenchymal transition (EMT) (e.g. EpCAM, a common marker for isolating CTCs). We also evaluated markers that describe immune suppression activity (PD-L1), EMT status (E-Cadherin, Vimentin, MUC1, Slug), as well as markers that can be therapeutically targeted/are indicative of resistance (e.g., p-YAP). For parallel immune profiling we assessed among others CD8/CD4 (T-cytotoxic/T-helpers respectively) and CD11b and HLA-DR (Myeloid-derived suppressor cells, MDSCs) expression. We found that p-YAP and MUC1 are significantly increased in CTCs detected in blood specimens from SCLC relapsed patients compared to treatment naïve patients. Both YAP and MUC1 have been shown to be play a role in therapy resistance and YAP inhibition is considered a promising therapeutic target. Our analysis also showed a significant increase of MDSC (CD33+CD11b+HLA-DR-) percentages and a decrease in the CD4/CD8 T cell ratio in the relapsed setting. Finally, when we used PHENOSTAMP, a previously developed reference EMT map for assessing EMT phenotypes in lung cancer clinical specimens at a personalized level, we observed an increase of EMT heterogeneity in CTCs in relapsed patients. Our data highlight the translational power of our approach in tracking therapy resistant cells during therapy directly in SCLC patient liquid biopsies towards assessing therapy resistance at a personalized level. Citation Format: Sayantan Bhattacharyya, Catherine A Stewart, Ashley M Victorian, Shafqat F Ehsan, Ester F Lujan, Alberto Duarte, John V Heymach, Jing Wang, Lauren A Byers, Loukia G Karacosta. Mapping therapy resistant phenotypes in SCLC liquid biopsies with single-cell proteomics at a personalized level [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B05.