Single-cell Pattern Research Articles

Conclusive evidence for the origin of the carcinoid component in strumal carcinoids and a new morphological pattern

Abstract Introduction/Objective There has been debate whether strumal carcinoids are carcinoids with areas resembling thyroid or arise from bonafide thyroid tissue in struma. While the thyroid nature has been proven by demonstrating positive TTF-1 in the struma component, evidence for the origin of the carcinoid component is sparse. Herein, we describe a case of strumal carcinoid and use a panel of markers to show definitive morphological and immunohistochemical evidence of the evolution of the neoplasm from struma ovarii to struma carcinoid, within the same lesion. We also report a unique component of a more sinister single cell pattern of invasion apart from the usual insular, trabecular, and mucinous patterns. Methods/Case Report A 40-year-old woman with an adnexal mass underwent bilateral salpingo- oophorectomy/hysterectomy. Microscopic analysis identified thyroid tissue consistent with struma ovarii, juxtaposed with a neuroendocrine neoplasm. The interface between the two showed strong PAX-8 and uniform TTF-1 staining in the struma but gradual loss of TTF-1 with retained/weak PAX-8 staining, moving away from the struma component and into the carcinoid. Additionally, a pattern of single cell invasion was identified, away from the main tumor and at the ovarian surface. These cells closely resemble luteinized ovarian stromal cells but are stained like the carcinoid tumor. Two months postoperatively, the patient had no evidence of recurrence or metastasis. Results (if a Case Study enter NA) NA Conclusion We have provided conclusive morphological and immunohistochemical evidence that strumal carcinoid develops from native neuroendocrine cells of thyroid tissue in struma ovarii. Specifically, retained/weak PAX-8 with interspersed TTF-1 in an adnexal carcinoid provides valuable insight when dealing with tumors of unknown etiology, strongly favoring an ovarian primary (specifically, from struma ovarii) and not metastasis from the gastrointestinal tract or other Mullerian tissue. Lastly, the single cell pattern of invasion that we elucidate here is something to be aware of in the context of metastases or frozen sections.

Single-cell multiplex immunocytochemistry in cell block preparations of metastatic breast cancer confirms sensitivity of GATA-binding protein 3 over gross cystic disease fluid protein 15 and mammaglobin.

Metastatic breast cancers are frequently encountered in cytology and require immunocytochemistry (ICC). In this study, traditional and multiplex ICC (mICC) for GATA-binding protein 3 (GATA3), gross cystic disease fluid protein 15 (GCDFP15), and mammaglobin (MMG) were performed with the aim of validating mICC in cell blocks, with further single-cell expression pattern analysis to identify the single markers and combinations of markers most sensitive in subtypes of breast cancer. GATA3, GCDFP15, and MMG were paired with OptiView 3,3'-diaminobenzidine and Ventana DISCOVERY Purple and Blue, respectively, with cyclical and serial staining. Bright-field imaging was performed with the Mantra 2 system and analyzed with the inForm Tissue Finder (Akoya Biosciences). Cell detection and phenotyping were further confirmed by two pathologists. In the 36 cases studied, traditional ICC and mICC demonstrated good concordance (kappa coefficient, >0.5; p<.01) at three cutoffs (1%, 5%, and 50%), except for GATA3 at the 1% cutoff. Single-marker positivity outnumbered double-marker positivity and the exceedingly rare triple-marker positivity (<3%). GATA3 was the leading single marker-positive phenotype in all breast cancer subtypes, except for MMG in estrogen receptor-positive, progesterone receptor-positive, and human epidermal growth factor receptor 2-positive (ER+/PR+/HER2+) breast cancers. Limited to two markers, GATA3/MMG included the greatest number of tumor cells for luminal breast cancers (ER+/PR+/HER2+, 60.6%; ER+/PR+/HER2+, 31.4%), whereas HER2-overexpressed breast cancers (27.4%) and triple-negative breast cancers (26.4%) favored the combination of GATA3/GCDFP15. For a single marker, GATA3 displayed the highest sensitivity. The addition of MMG for hormone receptor-positive breast cancers and GCDFP15 for hormone receptor-negative breast cancers further increased sensitivity. The low proportion of multimarker-positive cells suggested that the coexpression observed with traditional ICC is attributable to intratumoral heterogeneity, not genuine coexpression.

Prognostic value of micrometric substaging in pT1 bladder cancer patients treated with en-bloc transurethral resection.

We aimed to assess the oncological impact of micrometric extent of invasion in patients with pT1 bladder cancer (BCa) who underwent en-bloc resection for bladder tumour (ERBT). We retrospectively analysed the records and specimens of 106 pT1 high-grade BCa patients who underwent ERBT. The extent of invasion, such as depth from basal membrane, number of invasive foci, maximum width of invasive focus, muscularis mucosae invasion and infiltration pattern (pattern A: solid sheet-like, nodular or nested growth, pattern B: trabecular, small cluster or single-cell pattern) were evaluated by a single genitourinary pathologist. The end-points were recurrence-free (RFS) and progression-free survival (PFS). Within a median follow-up of 23 months, overall, 36 patients experienced recurrence and 13 patients experienced disease progression. The 2-year PFS differed significantly depending on depth from basal membrane (< 1.3 mm: 94.8% versus ≧ 1.3 mm: 65.2%, P = 0.005), maximum width of invasive focus (< 4 mm: 91.7% versus ≧ 4 mm: 62.3%, P < 0.001), muscularis mucosae (MM) invasion (above MM = 96.1% versus into or beyond MM = 64.8%, P = 0.002) and infiltration pattern (pattern A: 100% versus pattern B: 83.3%, P = 0.037). In a multivariable analysis, MM invasion [hazard ratio (HR) = 4.54, 95% confidence interval (CI) = 1.25-16.5] and maximum width of invasive focus ≧ 4 mm (HR = 4.79, 95% CI = 1.25-16.5) were independent prognostic factors of progression. En-bloc resection facilitates the evaluation of pathologic variables that might be useful in predicting disease recurrence and progression. In particular, not only the MM invasion but also the maximum width of invasion focus, reflecting the invasive volume, appear to be reliable prognosticators for disease progression.

Optimization of Semiconductor-Based SRR Metamaterials as Sensors

The development of hybrid sensor media is needed to achieve more efficient, sensitive, and accurate performance. Efforts to modify the structure of conventional metamaterials are carried out by integrating semiconductor materials which aim to improve the characteristics of optical properties, electrical properties, and sensitivity as sensors. This study aims to analyze and investigate changes in the optical properties of semiconductor-based metamaterials. The research was conducted through simulation and numerical methods to design and characterize the SRR metamaterial geometry, with a modified Nicolson-Ross-Weir approach, especially the optical parameters of refractive index. The single-cell square pattern SRR metamaterial geometry with a ring radius of 2.2 – 2.8 mm on quartz glass substrate designed at a smaller wavelength based on a maximum frequency source of 9 GHz. The square SRR metamaterial is integrated with several semiconductor materials such as silicon (Si), gallium arsenide (GaAs), and aluminum nitride (AlN). Changes in radius size cause a redshift with respect to radius enlargement. The increasing ring radius of SRR causes a higher resonance depth of the refractive index. Combining hybrid semiconductors with metamaterial results in more negative metamaterial properties as the refractive index becomes larger and negative. The addition of semiconductor material to the metamaterial substrate causes a negative refractive index to shift to a lower frequency.

Exploring NK-Cell molecules that impact the immune response and microenvironment in head and neck squamous cell carcinoma.

NK cells play a role in various cancers, but their role in head and neck squamous cell carcinoma (HNSCC) still needs to be explored. All public data are obtained from the Cancer Genome Atlas Program (TCGA) database. All analysis was performed using specific packages in R software. In our study, we quantified the immune microenvironment of HNSCC through multiple algorithms. Next, we identified NK cell-associated genes by quantifying NK cells, including SSNA1, TRIR, PAXX, DPP7, WDR34, EZR, PHLDA1 and ELOVL1. Then, we explored the single-cell expression pattern of these genes in the HNSCC microenvironment. Univariate Cox regression analysis indicated that the EZR, PHLDA1 and ELOVL1 were related to the prognosis of HNSCC patients. Following this, we selected EZR for further analysis. Our results showed that the patients with high EZR expression might have a poor prognosis and worse clinical features. Biological enrichment analysis showed that EZR is associated with many oncogenic pathways and a higher tumour stemness index. Meanwhile, we found that EZR can remodel the immune microenvironment of HNSCC. Moreover, we noticed that EZR could affect the immunotherapy and specific drug sensitivity, making it an underlying clinical target. In summary, our results can improve the understanding of NK cell in HNSCC. Meanwhile, we identified EZR as the underlying clinical target of HNSCC.

Integrative analysis illustrates the role of PCDH7 in lung cancer development, cisplatin resistance, and immunotherapy resistance: an underlying target.

Background: Cisplatin resistance is a common clinical problem in lung cancer. However, the underlying mechanisms have not yet been fully elucidated, highlighting the importance of searching for biological targets. Methods: Bioinformatics analysis is completed through downloaded public data (GSE21656, GSE108214, and TCGA) and specific R packages. The evaluation of cell proliferation ability is completed through CCK8 assay, colony formation, and EdU assay. The evaluation of cell invasion and migration ability is completed through transwell and wound-healing assays. In addition, we evaluated cell cisplatin sensitivity by calculating IC50. Results: Here, we found that PCDH7 may be involved in cisplatin resistance in lung cancer through public database analysis (GSE21656 and GSE108214). Then, a series of in vitro experiments was performed, which verified the cancer-promoting role of PCDH7 in NSCLC. Moreover, the results of IC50 detection showed that PCDH7 might be associated with cisplatin resistance of NSCLC. Next, we investigated the single-cell pattern, biological function, and immune analysis of PCDH7. Importantly, we noticed PCDH7 may regulate epithelial-mesenchymal transition activity, and the local infiltration of CD8+ T and activated NK cells. Furthermore, we noticed that patients with high PCDH7 expression might be more sensitive to bortezomib, docetaxel, and gemcitabine, and resistant to immunotherapy. Finally, a prognosis model based on three PCDH7-derived genes (GPX8, BCAR3, and TNS4) was constructed through a machine learning algorithm, which has good prediction ability on NSCLC patients' survival. Conclusion: Our research has improved the regulatory framework for cisplatin resistance in NSCLC and can provide direction for subsequent related research, especially regarding PCDH7.

Phenotypic Modulation of Adipose-Derived Stem Cells and Fibroblasts Treated with Povidone-Iodine and Chlorhexidine in Mono and Coculture Models.

Topical antiseptics are essential in wound treatment, and adipose-derived stem cells (ADSCs) have recently been proven to facilitate healing. However, the impact of antiseptics on ADSCs has not been fully elucidated, especially in relation to other relevant cell types present in the wound microenvironment, e.g., fibroblasts. This study evaluated the effects of chlorhexidine and povidone-iodine on four cellular constructs in 2D and 3D in vitro culture systems. Cell constructs were treated with two concentrations of each antiseptic, after which cell migration activity, α-SMA, and Ki67 marker expressions were assessed and compared. Both tested concentrations of povidone-iodine impaired migration and sprouting compared to chlorhexidine, which had minimal effects when used in low concentrations. The gap in the wound healing assay did not close after 24 h of povidone-iodine treatment, although, at the lower concentration, cells started to migrate in a single-cell movement pattern. Similarly, in 3D culture systems, sprouting with reduced spike formation was observed at high povidone-iodine concentrations. Both antiseptics modulated α-SMA and Ki67 marker expressions at 5 days following treatment. Although both antiseptics had cytotoxic effects dependent on drug concentration and cell type, povidone-iodine contributed more substantially to the healing process than chlorhexidine, acting especially on fibroblasts.

Diagnosis of thoracic SMARCA4-deficient undifferentiated tumor in cytology.

Although alterations in SMARCA4-deficient occur in non-small cell lung carcinoma (SD-NSCLC), thoracic SMARCA4-deficient undifferentiated tumor (TSDUT) is recognized as a distinct entity in the 2021 World Health Organization Classification of Thoracic Tumors because of unique morphologic, immunophenotypic and molecular features, and worse survival compared with SD-NSCLC. Cytologic diagnosis of TSDUT is clinically important because of its aggressive behavior and because it is often diagnosed by fine-needle aspiration because TSDUTs are usually unresectable at presentation. Here, we identify cytologic features that can be used for recognition of TSDUT and distinction from SD-NSCLC. Cytomorphologic features were investigated in cytology specimens from patients with TSDUT (n=11) and compared with a control group of patients with SD-NSCLC (n=20). The presence of classic rhabdoid morphology, at least focally, was entirely specific for TSDUT (n=6, 55%) compared with SD-NSCLC (n=0) in this study. TSDUT more frequently showed tumor necrosis (n=11, 100% vs. n=8, 40%; p=.001), dominant single-cell pattern on aspirate smears or touch preparation slides (n=8 [of 9], 80% vs. n=3, 15%; p=.010), nuclear molding (n=5, 45% vs. n=1, 5%; p=.013), and indistinct cell borders (n=11, 100% vs. n=5, 25%; P<.001) compared with SD-NSCLC, respectively. Cytomorphologic features occurring more frequently in TSDUT include tumor necrosis, dominant single-cell pattern, nuclear molding indistinct cell borders, and focal rhabdoid cells. Presence of these features in a cytology specimen of an undifferentiated tumor, particularly in a patient with a thoracic mass, should raise suspicion for TSDUT and prompt appropriate ancillary workup.

MP08-13 PROGNOSTIC VALUE OF MICROMETRIC SUBSTAGING IN pT1 BLADDER CANCER PATIENTS TREATED WITH EN BLOC TRANSURETHRAL RESECTION

You have accessJournal of UrologyCME1 Apr 2023MP08-13 PROGNOSTIC VALUE OF MICROMETRIC SUBSTAGING IN pT1 BLADDER CANCER PATIENTS TREATED WITH EN BLOC TRANSURETHRAL RESECTION Takafumi Yanagisawa, Shun Sato, Yasushi Hayashida, Yohei Okada, Akihiro Matsukawa, Kosuke Iwatani, Masayuki Shimoda, Hiroyuki Takahashi, Takahiro Kimura, Shahrokh F. Shariat, and Jun Miki Takafumi YanagisawaTakafumi Yanagisawa More articles by this author , Shun SatoShun Sato More articles by this author , Yasushi HayashidaYasushi Hayashida More articles by this author , Yohei OkadaYohei Okada More articles by this author , Akihiro MatsukawaAkihiro Matsukawa More articles by this author , Kosuke IwataniKosuke Iwatani More articles by this author , Masayuki ShimodaMasayuki Shimoda More articles by this author , Hiroyuki TakahashiHiroyuki Takahashi More articles by this author , Takahiro KimuraTakahiro Kimura More articles by this author , Shahrokh F. ShariatShahrokh F. Shariat More articles by this author , and Jun MikiJun Miki More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003223.13AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: We aimed to assess the oncologic impact of micrometric extent of invasion in patients with pT1 bladder cancer who underwent en bloc resection for bladder tumor (ERBT). METHODS: We retrospectively analyzed the records and specimens of 106 pT1 high-grade bladder cancer patients who underwent ERBT at multiple institutions. The extent of invasion such as depth from basal membrane, number of invasive foci, maximum width of invasive focus, muscularis mucosae invasion, and infiltration pattern were evaluated by a single genitourinary pathologist. For classification of infiltration pattern, we defined solid sheet-like, nodular or nested growth pattern as pattern A, and trabecular, small cluster or single cell pattern as pattern B. The endpoints were recurrence-free (RFS) and progression-free survival (PFS) stratified by invasion degrees. RESULTS: Overall, 24 patients (23%) had depth from basal membrane ≧1.3mm, 15 patients (14%) had maximum width of invasive focus ≧4mm, 32 patients (30%) had invasion into or beyond muscularis mucosae (MM), and 85 patients (80%) had infiltration pattern B. Within a median follow-up of 23 months, overall, 36 patients experienced disease recurrence and 13 patients experienced disease progression. The 2-year PFS significantly differed depending on depth from basal membrane (<1.3 mm: 94.8% vs. ≧1.3 mm: 65.2%, p=0.005), maximum width of invasive focus (<4 mm: 91.7% vs. ≧4 mm: 62.3%, p<0.001), MM invasion (above MM: 96.1% vs. into or beyond MM: 64.8%, p=0.002), and infiltration pattern (pattern A: 100% vs. pattern B: 83.3%, p=0.037). In a multivariable analysis that adjusted for the effects of standard predictors, MM invasion was an independent prognostic factor of progression (hazard ratio [HR]: 4.54, 95% confidence interval [CI]: 1.25-16.5). The maximum width of invasive focus ≧4mm was an independent prognostic factor of both recurrence (HR: 3.04, 95%CI: 1.40-6.58) and progression (HR: 4.79, 95%CI: 1.25-16.5). CONCLUSIONS: For T1 high-grade bladder cancer, MM invasion is a strong predictor of disease progression, while maximum width of invasion focus, reflecting the invasive volume, predicts both disease recurrence and progression. These two pathologic features could be used to further risk-stratify the heterogenous group of T1 bladder cancers. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e99 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takafumi Yanagisawa More articles by this author Shun Sato More articles by this author Yasushi Hayashida More articles by this author Yohei Okada More articles by this author Akihiro Matsukawa More articles by this author Kosuke Iwatani More articles by this author Masayuki Shimoda More articles by this author Hiroyuki Takahashi More articles by this author Takahiro Kimura More articles by this author Shahrokh F. Shariat More articles by this author Jun Miki More articles by this author Expand All Advertisement PDF downloadLoading ...

Buoyancy-Induced Instability of a Power-Law Fluid Saturating a Vertical Porous Slab

Abstract Many engineering applications involve porous media and rely on non-Newtonian working fluids. In this paper, the seepage flow of a non-Newtonian fluid saturating a vertical porous layer is studied. The buoyant flow is thermally driven by the boundaries of the porous layer, which are permeable surfaces kept at different temperatures. In order to model the seepage flow of both shear-thinning (pseudoplastic) and shear-thickening (dilatant) fluids, reference is made to the Ostwald-de Waele rheological model implemented via the power-law extended form of Darcy's law. The basic stationary flow is parallel to the vertical axis and shows a single-cell pattern, where the cell has infinite height and can display a core-region of enhanced/inhibited flow according to the fluid's rheological behavior. By applying small perturbations, a linear stability analysis of the basic flow is performed to determine the onset conditions for a multicellular pattern. This analysis is carried out numerically by employing the shooting method. The neutral stability curves and the values of the critical Rayleigh number are computed for different pseudoplastic and dilatant fluids. The behavior of a Newtonian fluid is also obtained as a limiting case.

Diagnostic distribution and pitfalls of glandular abnormalities in cervical cytology: a 25-year single-center study.

Detection of glandular abnormalities in Papanicolaou (Pap) tests is challenging. This study aimed to review our institute's experience interpreting such abnormalities, assess cytohistologic concordance, and identify cytomorphologic features associated with malignancy in follow-up histology. Patients with cytologically-detected glandular lesions identified in our pathology records from 1995 to 2020 were included in this study. Of the 683,197 Pap tests performed, 985 (0.144%) exhibited glandular abnormalities, 657 of which had tissue follow-up available. One hundred eighty-eight cases were cytologically interpreted as adenocarcinoma and histologically diagnosed as malignant tumors of various origins. There were 213 cases reported as atypical glandular cells (AGC) and nine cases as adenocarcinoma in cytology, yet they were found to be benign in follow-up histology. In addition, 48 cases diagnosed with AGC and six with adenocarcinoma cytology were found to have cervical squamous lesions in follow-up histology, including four squamous cell carcinomas. Among the cytomorphological features examined, nuclear membrane irregularity, three-dimensional clusters, single-cell pattern, and presence of mitoses were associated with malignant histology in follow-up. This study showed our institute's experience detecting glandular abnormalities in cervical cytology over a 25-year period, revealing the difficulty of this task. Nonetheless, the present study indicates that several cytological findings such as membrane irregularity, three-dimensional clusters, single-cell pattern, and evidence of proliferation could help distinguishing malignancy from a benign lesion.

Cytomorphologic Features of Metastatic Sarcomas in Body Cavity Fluids Overlaps with Other Common Malignancies

Background: Sarcomas rarely involve body cavity fluids, accounting for less than 5% of all malignant effusions. Most existing literature describes the cytomorphologic features of sarcomas in the setting of fine-needle aspiration and/or touch preparations of targeted tissue biopsies. The objective of this study was to investigate the morphologic spectrum displayed by metastatic sarcomas in body cavity fluid specimens. Methods: The pathology database at our institution was searched for sarcoma cases involving pleural, pericardial, and peritoneal fluids between 1994 and 2019. Their clinical presentations and cytopathologic features were reviewed. Immunohistochemical (IHC) stains were performed on the cellblock sections at our institution on the DAKO Autostainer (DAKO, Carpinteria, CA). Results: Nineteen cases were identified, including 14 females and 5 males, ranging in age from 4 to 88 years old (median: 28.6 years). The body cavity sites were 11 pleural, 7 peritoneal, and 1 pericardial. There were 5 rhabdomyosarcomas, 4 osteosarcomas, 4 Ewing sarcomas, 2 endometrial stromal sarcomas, 2 leiomyosarcomas, 1 clear cell sarcoma, and 1 liposarcoma. Malignant pleural effusion was the first presentation in only 2 cases (both alveolar rhabdomyosarcomas). Three different cytomorphologic patterns were seen: 1-glandular cell pattern showing large epithelioid cells with vacuolated cytoplasm (3 cases), a 2-pleomorphic single-cell pattern showing large highly atypical cells (6 cases); 3-small round blue cell pattern showing small to intermediate-sized dyscohesive cells with high NC ratio (10 cases). Conclusion: The cytomorphologic spectrum exhibited by metastatic sarcomas in body cavity fluids often overlaps with other common malignancies such as metastatic adenocarcinomas, poorly differentiated carcinomas, and lymphomas. Keywords: Sarcoma; Cytology; Peritoneal.

Serous cavity metastasis: Evaluation of unknown primary.

Malignant effusions can occur in patients with neoplasia. Once a metastatic diagnosis is confirmed, the primary site of origin of malignancy needs to be ascertained. This task can be challenging without a prior history of malignancy. In some patients their effusion may be the initial presentation of an underlying malignancy. Metastases usually present with a dual population of mesothelial and malignant cells. Combining cytomorphologic examination with ancillary testing such as immunocytochemistry can help identify the origin of the foreign malignant cell population. Helpful architectural clues include a single cell pattern, solid cell ball pattern, single file arrangement, papillary formation, psammoma bodies and background mucin. Useful cellular features include the presence of signet ring cells, small cells, pleomorphic and multinucleated giant cells, squamous cells, spindle cells and pigmentation. Rarely, despite an extensive work-up the primary site of origin for a malignant effusion may remain unresolved. This review article will be incorporated finally as one of the chapters in CMAS (CytoJournal Monograph/Atlas Series) #2. It is modified slightly from the chapter by the initial authors in the first edition of Cytopathologic Diagnosis of Serous Fluids.

Stability of a Buoyant Oldroyd-B Flow Saturating a Vertical Porous Layer with Open Boundaries

The performance of several engineering applications are strictly connected to the rheology of the working fluids and the Oldroyd-B model is widely employed to describe a linear viscoelastic behaviour. In the present paper, a buoyant Oldroyd-B flow in a vertical porous layer with permeable and isothermal boundaries is investigated. Seepage flow is modelled through an extended version of Darcy’s law which accounts for the Oldroyd-B rheology. The basic stationary flow is parallel to the vertical axis and describes a single-cell pattern where the cell has an infinite height. A linear stability analysis of such a basic flow is carried out to determine the onset conditions for a multicellular pattern. This analysis is performed numerically by employing the shooting method. The neutral stability curves and the values of the critical Rayleigh number are evaluated for different retardation time and relaxation time characteristics of the fluid. The study highlights the extent to which the viscoelasticity has a destabilising effect on the buoyant flow. For the limiting case of a Newtonian fluid, the known results available in the literature are recovered, namely a critical value of the Darcy–Rayleigh number equal to 197.081 and a corresponding critical wavenumber of 1.05950.

The Origin of the Cyathea delgadii Sternb. Somatic Embryos Is Determined by the Developmental State of Donor Tissue and Mutual Balance of Selected Metabolites.

Somatic embryogenesis is the formation of a plant embryo from a cell other than the product of gametic fusion. The need to recognize the determinants of somatic cell fate has prompted investigations on how endogenous factors of donor tissues can determine the pattern of somatic embryo origin. The undertaking of this study was enabled by the newly developed experimental system of somatic embryogenesis of the tree fern Cyathea delgadii Sternb., in which the embryos are produced in hormone-free medium. The contents of 89 endogenous compounds (such as sugars, auxins, cytokinins, gibberellins, stress-related hormones, phenolic acids, polyamines, and amino acids) and cytomorphological features were compared between two types of explants giving rise to somatic embryos of unicellular or multicellular origin. We found that a large content of maltose, 1-kestose, abscisic acid, biologically active gibberellins, and phenolic acids was characteristic for single-cell somatic embryo formation pattern. In contrast, high levels of starch, callose, kinetin riboside, arginine, and ethylene promoted their multicellular origin. Networks for visualization of the relations between studied compounds were constructed based on the data obtained from analyses of a Pearson correlation coefficient heatmap. Our findings present for the first time detailed features of donor tissue that can play an important role in the somatic-to-embryogenic transition and the somatic embryo origin.

Morphologic and immunohistochemical features of carcinoma involving microglandular adenosis of the breast following neoadjuvant chemotherapy.

Microglandular adenosis (MGA)-related lesions, including atypical MGA (AMGA) and carcinoma involving MGA (C-MGA), are characterized by epithelial atypia, negative hormone receptors and HER2 status, and can mimic invasive triple negative breast cancer (TNBC) in core needle biopsies (CNB) resulting in selection for treatment with neoadjuvant chemotherapy (NAC). We identified 12 cases of AMGA and/or C-MGA in post-NAC excision specimens (EXC) and analyzed their morphologic and immunohistochemical (IHC) features. All CNBs were initially diagnosed as containing TNBC. Upon re-review, TNBC was confirmed in 9 cases. In 3 CNBs AMGA and/or C-MGA had been interpreted as TNBC. AMGA was initially recognized in only 1 case but AMGA and/or C-MGA were present in an additional 9 CNBs. At EXC, no residual TNBC was present in 5 of 9 EXCs and all 12 cases showed residual AMGA and/or C-MGA. Similar to conventional MGA, AMGA and C-MGA were positive for S-100, laminin and collagen IV and negative for calponin and p63. Following NAC, these lesions retained their typical staining pattern despite acquiring treatment-related morphologic alterations, most notably of which were areas of single cell growth pattern seen in 8 EXCs. This study is the first to report the effects of NAC on AMGA and C-MGA. Our data showed no response of the AMGA and/or C-MGA following NAC in contrast to the high response rate of conventional TNBC. In particular, the infiltrative single cell pattern of post-NAC MGA-related lesions closely mimicked residual TNBC. The persistence of AMGA and C-MGA following NAC supports the notion that these lesions are distinct from conventional TNBC. Our findings also highlight the challenges in recognizing AMGA and C-MGA in CNBs which may lead to unwarranted treatment with NAC in the absence of conventional TNBC.

Abstract PO-35: Prognostic significance of MYC, BCL2, and BCL6 colocalization at single-cell resolution in DLBCL

Abstract MYC, BCL2, and BCL6 are commonly used markers for immunohistochemistry of Diffuse large B-cell lymphomas (DLBCL). Coexpression of MYC and BCL2 in particular constitutes a subgroup of “double expressor lymphomas” (DEL) with a distinct poor clinical outcome. However, it is not known if MYC and BCL2/BCL6 coexpression occurs in the same cell or in different cells within the tumor, as traditional immunohistochemistry (IHC) is limited by the number of markers that can be simultaneously assessed within formalin-fixed, paraffin-embedded (FFPE) samples. We set out to discover the clinical significance of MYC, BCL2, and BCL6 colocalization at single-cell resolution using multiplexed quantitative immunofluorescence (qIF) based on sequential OPAL-TSA staining and spectral microscopy on the Vectra platform. The initial discovery cohort comprised 90 cases of DLBCL from NUH Singapore with adequate clinical follow-up after R-CHOP therapy. We stratified each DLBCL tumor into 8 “clonal fractions” based on the possible permutations of MYC (M), BCL2 (2), and BCL6 (6) colocalization: M+2+6+, M+2+6-, M+2-6+, M+2-6-, M-2+6+, M-2-6+, M-2+6-, and M-2-6-. Interestingly, even within cases that fit traditional IHC criteria for “positivity” of MYC, BCL2, and BCL6, only a subset of cells within each case expressed multiple markers concurrently. Using the fraction of each of these clones as a continuous variable, Cox regression analysis revealed that the percentage of M+2+6- cells in a case was most predictive of poor survival. Importantly, the same clonal fraction (M+2+6-) was a significant poor prognostic feature in 2 smaller validation cohorts from SGH Singapore (n=41) and MD Anderson Cancer Centre USA (n=36). The single-cell staining pattern of these markers revealed a stark contrast between healthy tonsil tissue and DLBCL tissue. In the tonsil, colocalization of each marker was nonrandom (mutually exclusive BCL2 positivity in B cells outside the germinal center and BCL6 positivity inside the germinal center), whereas in DLBCL samples the mutual exclusivity pattern noted in the tonsil was lost, leading to a random distribution of colocalization of MYC, BCL2, and BCL6. The random nature of this colocalization allowed us to mathematically predict the “extent” of these 8 subclones from any data set with quantitative data of each single marker (MYC, BCL2, and BCL6). We therefore attempted to evaluate this model in RNA expression datasets of DLBCL cases with clinically annotated data. Remarkably, in concordance with our IF data, the “predicted” M+2+ 6- subgroup consistently was associated with an unfavorable prognosis in 3 independent mRNA datasets (GSE10846 n=233, GSE117556 n=469, GSE32918 n=140). In summary, we have for the first time assessed the expression of MYC, BCL2, and BCL6 at the single-cell level in DLBCL. These results may explain the apparent protective function of BCL6 expression in prior cohort studies of DEL, and provide a quantitative tool for the identification of DLBCL cases with poor survival on R-CHOP. Citation Format: Michal Hoppe, Shuangyi Fan, Patrick Jaynes, Phuong Mai Hoang, Liu Xin, Sanjay De Mel, Li Mei Poon, Esther Chan, Joanne Lee, Yen Lin Chee, Choon Kiat Ong, Tiffany Tang, Soon Thye Lim, Nicholas Francis Grigoropoulos, Sheng-Tsung Chang, Shih-Sung Chuang, Joseph Khoury, Hyungwon Choi, Wee Joo Chng, Siok-Bian Ng, Claudio Tripodo, Anand D. Jeyasekharan. Prognostic significance of MYC, BCL2, and BCL6 colocalization at single-cell resolution in DLBCL [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-35.

Clinicopathologic Study of Gleason Pattern 5 Prostatic Adenocarcinoma With "Single-cell" Growth Reveals 2 Distinct Types, One With "Plasmacytoid" Features.

Each Gleason score category of prostatic adenocarcinoma (or Grade Group) may encompass a diverse group of architectural patterns such as well-formed glands, poorly formed glands, cribriform structures, single cells, and/or solid sheets. We have noted heterogeneity within the single-cell subtype of Gleason pattern 5 prostatic adenocarcinoma that has not been fully addressed. Therefore, we retrospectively reviewed a series of radical prostatectomies with high-grade prostatic adenocarcinoma (Grade Group 4 or 5), identifying tumors with a component of single-cell infiltration. Additional cases identified prospectively were also included. TNM status, association with other histologic patterns, and clinical follow-up status were determined. Immunohistochemistry for NKX3.1, E-cadherin, p120 catenin, and prostate-specific antigen (PSA) were performed in each case. Eighteen cases with a component of well-developed Gleason pattern 5 characterized by single infiltrative cells that comprised ≥5% of the tumor were identified (15/202 retrospective radical prostatectomies with the high-grade disease [7.5%]). The single-cell pattern ranged from 5% to 50% of the tumor volume, with 5 cases containing ≥40%, and variable secondary architecture included diffuse infiltrating single cells with targetoid growth pattern around benign glands, solid expansive nests of noncohesive cells, and corded/single file growth pattern. Further morphologic analysis demonstrated 2 distinct histologic subtypes: (1) (subtype 1; n=9) monomorphic "plasmacytoid" tumor cells with eccentrically placed nuclei and variable intracytoplasmic vacuoles with bland cytology and discohesion and (2) (subtype 2; n=9) more cohesive tumor cells with greater cytologic atypia characterized by prominent nucleoli, greater variability in nuclear size/shape, occasional mitotic figures, and more irregular infiltration. By immunohistochemistry, NKX3.1 nuclear expression and PSA cytoplasmic expression was retained in all cases. Concomitant membranous E-cadherin loss and strong cytoplasmic p120 catenin expression were present in 5 of the 18 (28%) cases, all in subtype 1 (5/9, 56%). Overall, 56% (10/18) of patients had advanced-stage disease (≥pT3b), and 70% (7/10) of these patients had associated lymphovascular invasion. All patients had concomitant cribriform patterns of carcinoma. The outcome was available for 14 patients: 4 died of unknown cause; 6 had biochemical recurrence with distant bone metastasis in 5 of the 6; and 4 patients with <3 years of follow-up currently have undetectable serum PSA levels (2 patients received salvage radiotherapy with androgen deprivation and 2 remain on routine follow-up). In summary, the single-cell pattern of Gleason pattern 5 prostatic adenocarcinoma is uniformly associated with other high-risk histologic patterns (eg, cribriform growth), and high-stage disease with distant metastasis is not uncommon. Our data suggest that the "single-cell" Gleason pattern 5 prostatic adenocarcinoma contains 2 distinct subtypes. Somatic CDH1 alterations may play a role in the development of the "plasmacytoid" pattern characterized by monomorphic cytology with concomitant E-cadherin loss and aberrant p120 catenin expression.

Fine-needle aspiration cytomorphology of papillary thyroid carcinoma with NTRK gene rearrangement from a case series with predominantly indeterminate cytology.

Neurotrophic tyrosine kinase receptor (NTRK) rearrangement has been reported in a subset of papillary thyroid carcinoma (PTC) cases. Little is known about the cytomorphologic features of NTRK-rearranged PTC. We report an institutional series of 13 fine-needle aspiration (FNA) specimens of NTRK-rearranged PTC with a predominantly indeterminate cytology diagnosis. NTRK3 or NTRK1 rearrangements were detected on FNA or surgical specimens by next-generation sequencing. The 13 patients had a median age of 18 years; 10 patients were female and 3 patients were male. In 10 (77%) cases, cytology was indeterminate, and histopathologic follow-up was predominantly the follicular variant of PTC (n=8 [62%]), mostly infiltrative subtype. Of 12 FNA specimens available for review, a predominant loosely cohesive group pattern was the most commonly encountered architectural pattern (n=5 [41%]), followed by single cell (n=3 [25%]), thick cord (n=2 [17%]), and microfollicular pattern (n=2 [17%]). Background lymphocytic thyroiditis was observed in 9 cases. At the cellular level, the cytoplasm was moderate and granular, occasionally vacuolated. Classic PTC nuclear features (eg, nuclear enlargement, elongation, grooves, and nuclear membrane irregularity) were present but were often focal and subtle. Chromatin was often granular. Intranuclear pseudoinclusions were absent or rare. Our study demonstrates that most cases of NTRK rearrangement lack classic PTC cytomorphologic characteristics. Loosely cohesive groups and single cells with granular, sometimes vacuolated cytoplasm and subtle nuclear features are often seen on FNA specimens. Recognizing these characteristics may be helpful to preoperatively prompt molecular testing, including NTRK rearrangement analysis.

"Single-cell pattern" of adenocarcinoma cells in effusion cytology: Morphologic challenges of lung cancer.

Lung adenocarcinomas present as tight clusters and three-dimensional balls in effusion specimens. Unlike carcinomas of breast and stomach where singly lying malignant cells are seen in effusion samples, lung adenocarcinomas usually show cohesive morphology. This single-cell pattern may also be confused with reactive mesothelial cells. We studied the frequency of pulmonary adenocarcinoma with single-cell pattern cytomorphology in pleural effusion specimens. All cases reported as either suspicious or positive for malignancy on pleural effusion cytology (PFC) over the past 1 year were retrieved. The clinical details were obtained from requisition forms. Cases with predominant single-cell pattern, clinically suspicious of carcinoma lung were segregated. These were de-stained and immunocytochemistry (ICC) for TTF-1 was performed. Of 103 cases reported as either suspicious or positive for malignancy on PFC, 29 had a predominant single-cell pattern. Of these, 13 (44.8%) were primary lung carcinoma. The rest were metastasis from ovary (5; 17.2%), breast (2; 6.9%), stomach (2; 6.9%), lymphoma (1; 3.5%), and Ewing's sarcoma (1; 3.5%). Five (17.2%) were those with unknown primary. All cases of lung carcinoma were positive for TTF-1 ICC. Single-cell pattern of pulmonary adenocarcinoma is commoner than popularly believed. This pattern may be difficult to differentiate from carcinoma cells of other sites as well as from reactive mesothelial cells. A high degree of suspicion is therefore needed to perform relevant ICC to clinch the correct diagnosis.