Co-expression Of Markers Research Articles

Abstract 468: Quantitative assessment of IHC using computational pathology allows superior patient selection for biomarker-informed patients

Abstract Many targeted cancer therapies rely on biomarkers, which are assessed by standard pathologist scoring of immunohistochemically stained tissue. However, this process is subjective, semi-quantitative and does not assess expression heterogeneity. A quantitative method to measure IHC markers might therefore significantly improve patient selection particularly of proteins expressed at low levels. To address these challenges, we have developed the Quantitative Continuous Scoring (QCS) that deploys the power of fully supervised Deep Learning (DL) algorithms to provide objective and continuous data of biomarkers in digitized IHC whole slide images (WSI). The two DL-based algorithms, developed using pathologist input as the ground truth, identify areas of invasive tumor and segment each individual tumor cell across the WSI into pixels that represent cell nuclei, cytoplasm and/or membrane. This allows to compute biomarker expression as mean Optical Density (OD) in each of these subcellular compartments based on the Hue-Saturation-Density (HSD) model. Of note, this also allows computation of the spatial distribution of tumor cells across the WSI. The measured OD for each cell is aggregated as a histogram to quantitative continuous readouts for each patient sample. The method’s ability to accurately detect low expression range facilitates selection of antibody clones for IHC assays, has been successfully used to delineate mode of action and PK/PD mechanisms, has provided surrogate markers of spatial expression heterogeneity to predict potential bystander activity and has facilitated marker co-expression analysis to inform rational combination therapies. In retrospective clinical trials analysis, QCS showed superior performance in identifying a patient population gaining maximum treatment benefit. QCS-based quantification of PD-L1 membrane expression was able to stratify anti-PD-L1 treated late-stage non-small cell lung cancer (NSCLC) patients [NCT01693562] with a higher prevalence and more significant log rank p-value (64%, p=0.0001) for OS compared to pathologist TPS (59%, p=0.01). In summary, we describe a computational pathology-based approach for precise biomarker quantification and superior patient selection with broad applicability and the potential to transform pathology, thus addressing one of the key challenges of precision oncology. Citation Format: Hadassah Sade, Ansh Kapil, Philipp Wortmann, Andreas Spitzmueller, Nicolas Triltsch, Lina Meinecke, Susanne Haneder, Anatoliy Shumilov, Jan Lesniak, Valeria Bertani, Tze-Heng Tan, Ana Hidalgo-Sastre, Simon Christ, Andrea Storti, Regina Alleze, Dasa Medrikova, Jessica Chan, Simon Lanzmich, Markus Schick, Guenter Schmidt, J. Carl Barrett. Quantitative assessment of IHC using computational pathology allows superior patient selection for biomarker-informed patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 468.

Immunological microenvironment in colon cancer patients with different phenotypes of cancer stem cells.

e15503 Background: The purpose of this study was to analyze the effect of the CD44+ and CD133+ co-expression in cancer stem cells (CSCs) on lymphocytic microenvironment of colon cancer (CC). Methods: 200 CC patients received surgery as the first stage of treatment. The percentage of CSCs with the expression of CD44+ and/or CD133+ markers was studied in the tumor homogenates by flow cytometry, as well as some indicators of local immunity (CD3+, CD4+, CD8+, T regs (CD4+CD25+CD127dim), CD19+, PD-1, PD-L1, Th0, Tm, CD16/56+ and immunophenotypic characteristics of tumor cells (PD-L1, MHC-ABC). Results: Gradation depending on the absence or presence of co-expression of CSC markers on tumor cells allowed identification of 11 statistically significant differences out of 17 studied parameters of tumor cells and their lymphocytic microenvironment. Co-expression of CSC markers was accompanied by higher percentage of T regs (7.3±0.4 versus 5.3±0.5%), together with lower levels of CD4+ cells. At the same time, a higher content of the total number of T-lymphocytes was noted due to CD8+ with an increase in the percentage of memory T cells and a decline in naive T lymphocytes within this subpopulation. In addition, the co-expression of CSC markers was accompanied by a lower content of PD-L1 (34.3±3.0 vs. 42.9±2.5%) on lymphocytes and its higher content on tumor cells (10.6±1.5 vs. 4.1±0.8%), while the PD-1 expression on lymphocytes was higher (38.4±3.7 versus 22.3±2.9%). The presence of CD44+CD133+ CSCs was also accompanied by lower percentage of tumor cells expressing MHC class I (60.4±4.9 vs. 79.3±7.6%), which characterized the inhibition of recognition processes, and increased levels of CD8+, perhaps, should be considered as compensatory. Conclusions: The lymphocytic microenvironment of CC in the presence of CSCs with the CD44+CD133+ immunophenotype seems to be more immunosuppressive, according to the increase in the local content of T regs and the decrease in MHC-ABC expression. Higher expression of PD-L1 on tumor cells and PD-1 on lymphocytes allows activation of the PD-1/PD-L1 interaction, which enhances the immunosuppressive and growth-stimulating properties of the tumor microenvironment, but at the same time, makes tumor cells adequate targets for immunotherapy with immune checkpoint inhibitors.

Differential Single Cell Responses of Embryonic Stem Cells Versus Embryoid Bodies to Gravity Mechanostimulation

The forces generated by gravity have shaped life on Earth and impact gene expression and morphogenesis during early development. Conversely, disuse on Earth or during spaceflight, reduces normal mechanical loading of organisms, resulting in altered cell and tissue function. Although gravity mechanical loading in adult mammals is known to promote increased cell proliferation and differentiation, little is known about how distinct cell types respond to gravity mechanostimulation during early development. In this study we sought to understand, with single cell RNA-sequencing resolution, how a 60-min pulse of 50 g hypergravity (HG)/5 kPa hydrostatic pressure, influences transcriptomic regulation of developmental processes in the embryoid body (EB) model. Our study included both day-9 EBs and progenitor mouse embryonic stem cells (ESCs) with or without the HG pulse. Single cell t-distributed stochastic neighbor mapping shows limited transcriptome shifts in response to the HG pulse in either ESCs or EBs; this pulse however, induces greater positional shifts in EB mapping compared to ESCs, indicating the influence of mechanotransduction is more pronounced in later states of cell commitment within the developmental program. More specifically, HG resulted in upregulation of self-renewal and angiogenesis genes in ESCs, while in EBs, HG loading was associated with upregulation of Gene Ontology-pathways for multicellular development, mechanical signal transduction, and DNA damage repair. Cluster transcriptome analysis of the EBs show HG promotes maintenance of transitory cell phenotypes in early development; including EB cluster co-expression of markers for progenitor, post-implant epiblast, and primitive endoderm phenotypes with HG pulse but expression exclusivity in the non-pulsed clusters. Pseudotime analysis identified three branching cell types susceptible to HG induction of cell fate decisions. In totality, this study provides novel evidence that ESC maintenance and EB development can be regulated by gravity mechanostimulation and that stem cells committed to a differentiation program are more sensitive to gravity-induced changes to their transcriptome.

Characterization of HIV-1 Infection in Microglia-Containing Human Cerebral Organoids.

The achievement of an HIV cure is dependent on the eradication or permanent silencing of HIV-latent viral reservoirs, including the understudied central nervous system (CNS) reservoir. This requires a deep understanding of the molecular mechanisms of HIV’s entry into the CNS, latency establishment, persistence, and reversal. Therefore, representative CNS culture models that reflect the intercellular dynamics and pathophysiology of the human brain are urgently needed in order to study the CNS viral reservoir and HIV-induced neuropathogenesis. In this study, we characterized a human cerebral organoid model in which microglia grow intrinsically as a CNS culture model to study HIV infection in the CNS. We demonstrated that both cerebral organoids and isolated organoid-derived microglia (oMG), infected with replication-competent HIVbal reporter viruses, support productive HIV infection via the CCR5 co-receptor. Productive HIV infection was only observed in microglial cells. Fluorescence analysis revealed microglia as the only HIV target cell. Susceptibility to HIV infection was dependent on the co-expression of microglia-specific markers and the CD4 and CCR5 HIV receptors. Altogether, this model will be a valuable tool within the HIV research community to study HIV–CNS interactions, the underlying mechanisms of HIV-associated neurological disorders (HAND), and the efficacy of new therapeutic and curative strategies on the CNS viral reservoir.

A comparative transcriptomic analysis of glucagon-like peptide-1 receptor- and glucose-dependent insulinotropic polypeptide receptor-expressing cells in the hypothalamus

ObjectiveThe hypothalamus is a key region of the brain implicated in homeostatic regulation, and is an integral centre for the control of feeding behaviour. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones with potent glucoregulatory function through engagement of their respective cognate receptors, GLP-1R and GIPR. Recent evidence indicates that there is a synergistic effect of combining GIP- and GLP-1-based pharmacology on appetite and body weight. The mechanisms underlying the enhanced weight loss exhibited by GIPR/GLP-1R co-agonism are unknown. Gipr and Glp1r are expressed in the hypothalamus in both rodents and humans. To better understand incretin receptor-expressing cell populations, we compared the cell types and expression profiles of Gipr- and Glp1r-expressing hypothalamic cells using single-cell RNA sequencing. MethodsUsing Glp1r-Cre or Gipr-Cre transgenic mouse lines, fluorescent reporters were introduced into either Glp1r- or Gipr-expressing cells, respectively, upon crossing with a ROSA26-EYFP reporter strain. From the hypothalami of these mice, fluorescent Glp1rEYFP+ or GiprEYFP+ cells were FACS-purified and sequenced using single-cell RNA sequencing. Transcriptomic analysis provided a survey of both non-neuronal and neuronal cells, and comparisons between Glp1rEYFP+ and GiprEYFP + populations were made. ResultsA total of 14,091 Glp1rEYFP+ and GiprEYFP+ cells were isolated, sequenced and taken forward for bioinformatic analysis. Both Glp1rEYFP+ and GiprEYFP+ hypothalamic populations were transcriptomically highly heterogeneous, representing vascular cell types, oligodendrocytes, astrocytes, microglia, and neurons. The majority of GiprEYFP+ cells were non-neuronal, whereas the Glp1rEYFP+ population was evenly split between neuronal and non-neuronal cell types. Both Glp1rEYFP+ and GiprEYFP+ oligodendrocytes express markers for mature, myelin-forming oligodendrocytes. While mural cells are represented in both Glp1rEYFP+ and GiprEYFP+ populations, Glp1rEYFP+ mural cells are largely smooth muscle cells, while the majority of GiprEYFP+ mural cells are pericytes. The co-expression of regional markers indicate that clusters of Glp1rEYFP+ and GiprEYFP+ neurons have been isolated from the arcuate, ventromedial, lateral, tuberal, suprachiasmatic, and premammillary nuclei of the hypothalamus. ConclusionsWe have provided a detailed comparison of Glp1r and Gipr cells of the hypothalamus with single-cell resolution. This resource will provide mechanistic insight into how engaging Gipr- and Glp1r-expressing cells of the hypothalamus may result in changes in feeding behaviour and energy balance.

Donor T and NK Cells with a Special Tissue-Resident Memory Phenotype Migrate into the Periphery of Lung Transplant Recipients - A Potential Feature for Tolerance Development

<h3>Purpose</h3> After lung transplantation (LuTx), a transient chimerism of donor cells can be observed in recipient blood due to the migration of lymphocytes from the transplanted lung into the periphery. We aimed to characterize the phenotype of donor T and NK cells and to investigate their potential origin from tissue-resident memory (TRM) T and NK cells. <h3>Methods</h3> Lymphocyte dynamics in recipient blood were determined in n=97 LuTx patients directly (T0), 24 hours (T24) and 3 (wks) weeks after LuTx using flow cytometry. Donor T and NK cells were analyzed by HLA class I allele-specific mAb in n=44 LuTx recipients. T and NK cell markers were used to discriminate circulating from TMR subsets in blood, organ storage solutions (perfusates, n=97), explanted lung parenchyma (n=28) and donor trachea (n=17). Single cell mRNA sequencing of explant lung parenchyma was conducted (n=16). <h3>Results</h3> In peripheral blood of all recipients, donor-derived T and NK cells were detected at T0, T24 and 3 wks after LuTx, showed higher CD69 expression compared to recipient cells (p=0.01 to 0.04) and were mostly CCR7⁻ memory cells. This phenotype was similar to T and NK cells in corresponding perfusates. In recipient parenchyma and donor trachea, most CD69⁺ T and NK cells showed coexpression of TRM markers (CD103, CD49a, PD-1) with significant enrichment in trachea (p<0.05). These other TRM markers were not found in circulating donor T and NK cells or in perfusates, arguing for different memory T and NK cell subsets. Sc-mRNA sequencing confirmed distinct TRM and TRM-like T cell subsets in lung parenchyma according to separate expression profiles. The presence of TRM T and NK cells did not have an impact on primary graft dysfunction (PGD) 24h after LuTx. Yet, patients with high frequencies of donor T cells showed a trend towards CLAD-free survival 2 years post LuTx, although no statistical significance was reached (p=0.15). <h3>Conclusion</h3> Our results demonstrate that donor T and NK cells found in recipient blood immediately after LuTx are separate subsets from circulating as well as pulmonary TRM T and NK cells, since they express CD69 but lack expression of other classical TRM markers. Donor T cells may be clinically relevant for tolerance induction and long-term survival after transplantation due to their unique features.

Desmoplastic Small Round Cell Tumor Is an Important Mimic of High-Grade Carcinoma in Colonic Biopsy Samples

Abstract Desmoplastic small round cell tumor (DSRCT) is a rare but aggressive malignancy that usually occurs within the intra-abdominal cavities of young adult males. Most DSRCTs can be reliably diagnosed based on the presence of characteristic morphology; coexpression of epithelial, myogenic, and neural markers; and detection of the reciprocal translocation t(11;22)(p13:21) associated with the EWS::WT1 fusion. However, occasional tumors lack classic features, affect older adults, or are located outside the abdominal cavity. Mucosal biopsy samples can pose challenges when both the stromal and epithelial components are not represented. Herein, we present a case of DSRCT that simulated clinical and histologic features of a colonic adenocarcinoma. Biopsy sampling revealed nests of cytokeratin-positive epithelioid cells that simulated a high-grade carcinoma with neuroendocrine features. This case report emphasizes the importance of considering this rare entity when presented with high-grade epithelioid tumors that occur in young patients.

Epithelioid Sarcoma: An overview with emphasis on its differentiation from morphologic mimics

Epithelioid sarcoma (ES) is a malignant mesenchymal neoplasm that exhibits epithelioid cytomorphology and a predominantly epithelial phenotype. The principal types based on histopathological features and location; include the classic or conventional type (CES) and the proximal type (PES). The aim of the study was to analyze our cases of ES regarding clinical, pathological and immunohistochemical features.This is a retrospective study. Seven cases of ES were included in the study. Clinical and pathological details were retrieved from patient records. Details like site, size, histopathological type and IHC features of the tumors were studied. Out of seven, five were PES and two were CES. There were six males and one female; with age ranging from 12 to 68 years. Total five tumors involved extremities. One was noted at a rare site, paratesticular region. Three tumors were larger than 5 cm in greatest dimension. On immunohistochemistry (IHC), all the tumors were reactive for CK, EMA, Vimentin, Ca 125 and showed loss of nuclear expression of INI1. CD 34 was expressed in 6 out of 7 cases.ES is a rare aggressive malignant tumor with dismal prognosis. It is often misdiagnosed because of nonspecific clinical features at presentation. Helpful clues in diagnosis are tumors in young males with epithelioid and/or spindle cell morphology, Rhabdoid cells and granuloma like central necrosis. Co-expression of epithelial and mesenchymal markers along with reactivity for CD34 and Ca 125 and loss on INI1 expression on IHC substantiate the diagnosis of ES.

Co-expression of cancer stem cell markers, SALL4/ALDH1A1, is associated with tumor aggressiveness and poor survival in patients with serous ovarian carcinoma

BackgroundSpalt-like transcription factor 4 (SALL4) and aldehyde dehydrogenase1 family member A1 (ALDH1A1) expressing cells have been characterized as possessing stem cell-like properties known as cancer stem cell marker in serous ovarian carcinoma (SOC).MethodsThe association between SALL4 and ALDH1A1 was observed based on literature review and bioinformatics tools. Therefore, this study aimed to investigate the association between the co-expression of SALL4/ALDH1A1 proteins and clinicopathological parameters and their prognostic value in SOC patients using immunohistochemical staining on tissue microarrays (TMAs). Furthermore, benign tumors and normal tissue samples were compared with the expression of the tumor tissue samples.ResultsIncreased co-expression of SALL4/ALDH1A1 was found to be significantly associated with the advanced FIGO stage (P = 0.047), and distant metastasis (P = 0.028). The results of Kaplan–Meier survival analysis indicated significant differences between disease- specific survival (DSS; P = 0.034) or progression-free survival (PFS; P = 0.018) and the patients with high and low co-expression of SALL4/ALDH1A1, respectively. Furthermore, high level co-expression of SALL4/ALDH1A1 was a significant predictor of worse DSS and PFS in the univariate analysis. The data also indicated that the co-expression of SALL4/ALDH1A1 was an independent prognostic factor affecting PFS. Moreover, the co-expression of SALL4/ALDH1A1 added prognostic values of DSS in patients with SOC who had grade III versus grade I in multivariate analysis.ConclusionsOur data demonstrated that high co-expression of SALL4/ALDH1A1 was found to be significantly associated with tumor aggressiveness and worse DSS or PFS in SOC patients. Therefore, co-expression of SALL4/ALDH1A1 may serve as a potential prognostic biomarker of cancer progression in these cases.

Reduced Zeb1 Expression in Prostate Cancer Cells Leads to an Aggressive Partial-EMT Phenotype Associated with Altered Global Methylation Patterns.

Prostate cancer is the most common cancer in American men and the second leading cause of cancer-related death. Most of these deaths are associated with metastasis, a process involving the epithelial-to-mesenchymal (EMT) transition. Furthermore, growing evidence suggests that partial-EMT (p-EMT) may lead to more aggressive disease than complete EMT. In this study, the EMT-inducing transcription factor Zeb1 was knocked down in mesenchymal PC-3 prostate cancer cells (Zeb1KD) and resulting changes in cellular phenotype were assessed using protein and RNA analysis, invasion and migration assays, cell morphology assays, and DNA methylation chip analysis. Inducible knockdown of Zeb1 resulted in a p-EMT phenotype including co-expression of epithelial and mesenchymal markers, a mixed epithelial/mesenchymal morphology, increased invasion and migration, and enhanced expression of p-EMT markers relative to PC-3 mesenchymal controls (p ≤ 0.05). Treatment of Zeb1KD cells with the global de-methylating drug 5-azacytidine (5-aza) mitigated the observed aggressive p-EMT phenotype (p ≤ 0.05). DNA methylation chip analysis revealed 10 potential targets for identifying and/or targeting aggressive p-EMT prostate cancer in the future. These findings provide a framework to enhance prognostic and/or therapeutic options for aggressive prostate cancer in the future by identifying new p-EMT biomarkers to classify patients with aggressive disease who may benefit from 5-aza treatment.

Biomarker Analysis of a Phase Ia/Ib Open-Label, Multicentre Study of Tiragolumab or Tiragolumab + Rituximab in Patients with Relapsed or Refractory (R/R) B-Cell Non-Hodgkin Lymphoma (NHL)

Background: NHL is the most common hematologic malignancy in adults, with follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL) being the most common subtypes. Despite therapeutic advances, most patients will experience relapse. New treatments are therefore needed to improve the outcome of patients with R/R NHL. T cell immunoreceptor with Ig and ITIM domains (TIGIT) is a well-known immune inhibitory receptor expressed on the surface of activated T cell and natural killer (NK)-cell subsets. TIGIT is expressed at higher levels than other checkpoints in intratumoral T cells in NHL and is highly correlated with PD-1 expression and T cell infiltration. This phase Ia/Ib trial (NCT04045028) evaluated the safety and pharmacokinetics of the anti-TIGIT agent, tiragolumab, alone or in combination with rituximab.Methods: Patients were recruited with histologically confirmed B-cell NHL whose disease has relapsed or failed to respond to ≥2 prior systemic treatment regimens, had ECOG PS 0-1, adequate hematologic and end organ function, and no history of CNS lymphoma. Patients received tiragolumab 600 mg IV Q3W with or without rituximab 375 mg/m 2 IV for the initial dose and 1400 mg SC rituximab/23400 U rHuPH20 QW for 8 doses. Here, we evaluate biomarker data collected from patients with R/R NHL dosed with tiragolumab as a single agent or in combination with rituximab via flow cytometry and IHC.Results: At data cut-off (July 2021), biomarker data had been collected from 14 patients with NHL. Baseline CD8 T cell density within the tumor region evaluated via IHC for these patients was between 500-6000 per mmA 2. In the peripheral blood of the 7 patients dosed with the combination of tiragolumab and rituximab, CD8 T cell expansion observed via absolute counts by flow cytometry was seen in 2 patients. Among the 7 patients, NK/NKT CD25 expression remained unchanged and a modest increase in NK CD69 expression was sustained above baseline in 1 patient. Overall, transient NK cell activation via increased CD69 expression was observed in 2 patients, which would be expected from the addition of rituximab. Increased PD-L1 expression was observed on multiple lymphocyte subsets in 4 of 7 patients in this cohort.Of the 7 patients who received single agent tiragolumab, trends in increased CD69 expression on NKs were observed in 4 patients and NK/NKT CD25 expression in 3 patients. A modest CD8 T cell activation, via increased CD69 expression, was observed in 2 patients, though T cell counts remained unchanged. At baseline, TIGIT was abundantly expressed on peripheral blood CD8 T cells, while co-expression of exhaustion markers on CD8 T cells was less widely observed.Although one patient experienced a sustained response, no other patients achieved clinical benefit. This heavily pretreated 65-year-old female patient with FL had an objective partial response (best overall response), determined via Lugano criteria, with a response duration on single agent tiragolumab for 11 months. The patient had a two-fold upregulated CD69 expression on NKs and sixty-three-fold CD25 upregulated expression on NK/NKTs, as well as increased frequencies of PD-L1+ on immune cells over course of treatment. In this patient, relatively higher TIGIT and lower expression of exhaustion markers on CD8 T cells were observed at baseline and over treatment compared to other patients analyzed.Conclusions: In this study, tiragolumab as a single agent and in combination with rituximab was seen to result in increased PD-L1 expression on multiple lymphocyte subsets (including B cells, CD4/CD8 T cells, and NKs), which support the combination of tiragolumab with PD-L1/PD-1 inhibitors. Increases in NK/NKT CD25 expression could suggest a tiragolumab-mediated increase in proliferative potential but further investigations are needed to confirm. A patient with R/R FL in this study was observed to have the first documented objective response to single agent tiragolumab in this disease indication, suggesting biomarker-driven combination strategies may be important in this population. DisclosuresRuppert: Genentech, Inc.: Current Employment. Cuchelkar: Genentech, Inc.: Current Employment. Meng: Genentech, Inc.: Current Employment. Cho: Genentech, Inc.: Current Employment; F. Hoffmann La Roche, Ltd: Current holder of individual stocks in a privately-held company. Lear: Genentech, Inc.: Current Employment. Wong: Genentech, Inc.: Current Employment. Raval: Genentech, Inc.: Ended employment in the past 24 months; Arcus Bioscience: Current Employment, Current holder of individual stocks in a privately-held company. Nouet: F. Hoffmann La Roche, Ltd: Current Employment.

Genetic Diversity within Leukemia-Associated Immunophenotype-Defined Subclones in AML

Acute myeloid leukemia (AML) is a heterogeneous clonal disease and in different patients, different combinations of mutations may be found that drive the disease. Also within patients, various leukemic clones with a different combination of mutations may co-exist, reflecting various evolutionary stages of the disease. At present, flowcytometry and mutational analyses are used to keep track of the malignant cells during and after treatment. Using panels of antibodies, abnormal cells can be identified when lineage markers are over- or underexpressed, or when abnormal differentiation leads to the co-expression of markers that normally are not expressed together on hematopoietic cells. These patient-specific leukemia-associated immunophenptypes (LAIPs) can be followed during and after treatment, and enable the early detection of resistant and recurring disease. Often, several LAIPs are present simultaneously within a patient, but their clonal relationship is not always clear. In addition, next-generation sequencing and mutation-specific PCR can be used to follow the leukemic cells. As the clinical consequences of MRD-positivity may consist of high-dose re-induction chemotherapy or stem cell transplantation, accurate results are of the highest importance. Although it has already been shown that flowcytometry and mutational analysis may have added value, in-depth information on the genetic make-up of immunophenotypically defined normal and leukemic subclones and their clonal relationship in AML is still scarce. To study the genetic similarities and differences between various normal and leukemic subclones defined by immunophenotyping, we performed an in-depth analysis of the genetic makeup of FACS-sorted immunophenotypically normal and aberrant subclones in 10 cases of AML. In total, 86 LAIPs and normal cell fractions were sorted and fully genotyped using error-corrected sequencing with a panel genes that are recurrently mutated in AML. In some cases, different aberrant populations from the same patient carried identical mutations, indicating that the leukemia was highly homogeneous, and that the phenotypically different LAIPs all were part of the same genetic clone. In these cases, monitoring the disease by following one of the present LAIPs would be sufficient, or, alternatively, following disease dynamics by molecularly monitoring one of the mutations would accurately show the dynamics of the disease. In other cases, different subclones harbored a different set of mutations. In addition, immunophenotypically normal populations frequently carried mutations, or, inversely, no mutations were found in immunophenotypically clearly aberrant clones. In these cases, following the disease by LAIPs or molecular markers would carry the risk that several subclones would be missed. In case these clones would be relevant for resistance or relapse, using either technique would be at risk to fail to detect MRD positivity. This work underscores that currently available, state-of-the-art immunophotyping and gene panels have added value and should be used simultaneously. We conclude that currently, MRD detection by molecular and immunophenotypic techniques are complementary, and should be used in parallel in order to obtain the most complete view on resistant disease and early detection of relapse. At the same time, both techniques can be further optimized with more monoclonal antibodies and genes that are screened for mutations, in order to increase their resolution. DisclosuresNo relevant conflicts of interest to declare.

Cytomorphologic findings of thyroid carcinoma showing thymus-like (CASTLE) differentiation: A case report

Abstract Introduction/Objective Thyroid carcinoma showing thymus-like differentiation also known as intrathyroidal thymic carcinoma is a rare thyroid neoplasm arising from either an ectopic intrathyroidal thymic tissue or from remnants of thymobranchial pouches. On fine needle aspiration (FNA) its diagnosis can be challenging due to overlapping morphologic features with other aggressive thyroid carcinomas. Methods/Case Report A 31-year-old female consulted for 6-month history of neck swelling and tenderness. Thyroid ultrasound demonstrated a 2.6 x 2.5 x 2.1 cm nodule in the right lobe with punctate calcifications. FNA showed cellular smears composed of loosely cohesive and single basaloid neoplastic cells exhibiting significant cellular and nuclear pleomorphism. Focal squamous differentiation with keratinization was noted on the cell block sections. Immunocytochemical stains showed that the tumor was positive for cytokeratin AE1/AE3, p40, p63, CD117 and CD5 consistent with intrathyroidal thymic carcinoma. The Ki67 proliferative index was approximately 40%. A right thyroidectomy with central neck dissection confirmed the diagnosis and showed a 3.0 cm tumor with invasion into skeletal muscle, lymphovascular invasion and positive lymph nodes. The patient completed adjuvant radiotherapy and remain in remission at 3-months follow-up. Results (if a Case Study enter NA) NA Conclusion Intrathyroidal thymic carcinoma is a rare thyroid neoplasm that frequently shows squamous differentiation and therefore overlaps with papillary thyroid carcinoma with squamous morulae, squamous cell carcinoma and anaplastic carcinoma. The coexpression of squamous markers together with CD5 and CD117 allows the recognition of CASTLE on FNA samples.

Oviductal Telocytes in Patients with Uterine Myoma.

Tubal factor infertility occurs in 30–35% of infertile pairs and may be caused by impaired muscular contractility and ciliary beating as well as immunological imbalance and chronic inflammation. Newly discovered telocytes (TCs) have a wide palette of features, which play a role in oviduct physiology. We have observed tissue samples from human fallopian tubes in patients with and without uterine myoma by immunolabelling. According to the immunohistochemical co-expression of markers, it has been determined that TCs are engaged in a wide range of physiological processes, including local innervation, sensitivity to hypoxia, regulation of calcium, and sex steroid hormones balances. Due to the proximity of NOS- and ChAT-positive nerve fibers and the expression of ion channels markers, tubal TCs might be considered conductor cells. Additionally, their integration in contractions and cilia physiology in the context of fertility has been revealed. We have observed the difference in telocytes expression in the human oviduct between groups of patients and attempted to describe this population of cells specifically in the case of infertility development, a clinically relevant avenue for further studies.

Neutrophil Extracellular Traps Induce the Epithelial-Mesenchymal Transition: Implications in Post-COVID-19 Fibrosis

The release of neutrophil extracellular traps (NETs), a process termed NETosis, avoids pathogen spread but may cause tissue injury. NETs have been found in severe COVID-19 patients, but their role in disease development is still unknown. The aim of this study is to assess the capacity of NETs to drive epithelial-mesenchymal transition (EMT) of lung epithelial cells and to analyze the involvement of NETs in COVID-19. Bronchoalveolar lavage fluid of severe COVID-19 patients showed high concentration of NETs that correlates with neutrophils count; moreover, the analysis of lung tissues of COVID-19 deceased patients showed a subset of alveolar reactive pneumocytes with a co-expression of epithelial marker and a mesenchymal marker, confirming the induction of EMT mechanism after severe SARS-CoV2 infection. By airway in vitro models, cultivating A549 or 16HBE at air-liquid interface, adding alveolar macrophages (AM), neutrophils and SARS-CoV2, we demonstrated that to trigger a complete EMT expression pattern are necessary the induction of NETosis by SARS-CoV2 and the secretion of AM factors (TGF-β, IL8 and IL1β). All our results highlight the possible mechanism that can induce lung fibrosis after SARS-CoV2 infection.

Monocyte and macrophage derived myofibroblasts: Is it fate? A review of the current evidence.

Since the discovery of the myofibroblast over 50 years ago, much has been learned about its role in wound healing and fibrosis. Its origin, however, remains controversial, with a number of progenitor cells being proposed. Macrophage-myofibroblast transition (MMT) is a recent term coined in 2014 that describes the mechanism through which macrophages, derived from circulating monocytes originating in the bone marrow, transformed into myofibroblasts and contributed to kidney fibrosis. Over the past years, several studies have confirmed the existence of MMT in various systems, suggesting that MMT could potentially occur in all fibrotic conditions and constitute a reasonable therapeutic target to prevent progressive fibrotic disease. In this perspective, we examined recent evidence supporting the notion of MMT in both human disease and experimental models across organ systems. Mechanistic insight from these studies and information from in vitro studies is provided. The findings substantiating plausible MMT showcased the co-expression of macrophage and myofibroblast markers, including CD68 or F4/80 (macrophage) and α-SMA (myofibroblast), in fibroblast-like cells. Furthermore, fate-mapping experiments in murine models exhibiting myeloid-derived myofibroblasts in the tissue further provide direct evidence for MMT. Additionally, we provide some evidence from single cell RNA sequencing experiments confirmed by fluorescent in situ hybridisation studies, showing monocyte/macrophage and myofibroblast markers co-expressed in lung tissue from patients with fibrotic lung disease. In conclusion, MMT is likely a significant contributor to myofibroblast formation in wound healing and fibrotic disease across organ systems. Circulating precursors including monocytes and the molecular mechanisms governing MMT could constitute valid targets and provide insight for the development of novel antifibrotic therapies; however, further understanding of these processes is warranted.

FAK inhibition alone or in combination with adjuvant therapies reduces cancer stem cell activity

Cancer stem-like cells (CSC) contribute to therapy resistance and recurrence. Focal adhesion kinase (FAK) has a role in CSC regulation. We determined the effect of FAK inhibition on breast CSC activity alone and in combination with adjuvant therapies. FAK inhibition reduced CSC activity and self-renewal across all molecular subtypes in primary human breast cancer samples. Combined FAK and paclitaxel reduced self-renewal in triple negative cell lines. An invasive breast cancer cohort confirmed high FAK expression correlated with increased risk of recurrence and reduced survival. Co-expression of FAK and CSC markers was associated with the poorest prognosis, identifying a high-risk patient population. Combined FAK and paclitaxel treatment reduced tumour size, Ki67, ex-vivo mammospheres and ALDH+ expression in two triple negative patient derived Xenograft (PDX) models. Combined treatment reduced tumour initiation in a limiting dilution re-implantation PDX model. Combined FAK inhibition with adjuvant therapy has the potential to improve breast cancer survival.

NEBULA is a fast negative binomial mixed model for differential or co-expression analysis of large-scale multi-subject single-cell data

The increasing availability of single-cell data revolutionizes the understanding of biological mechanisms at cellular resolution. For differential expression analysis in multi-subject single-cell data, negative binomial mixed models account for both subject-level and cell-level overdispersions, but are computationally demanding. Here, we propose an efficient NEgative Binomial mixed model Using a Large-sample Approximation (NEBULA). The speed gain is achieved by analytically solving high-dimensional integrals instead of using the Laplace approximation. We demonstrate that NEBULA is orders of magnitude faster than existing tools and controls false-positive errors in marker gene identification and co-expression analysis. Using NEBULA in Alzheimer’s disease cohort data sets, we found that the cell-level expression of APOE correlated with that of other genetic risk factors (including CLU, CST3, TREM2, C1q, and ITM2B) in a cell-type-specific pattern and an isoform-dependent manner in microglia. NEBULA opens up a new avenue for the broad application of mixed models to large-scale multi-subject single-cell data.

Immune contexture in high-risk soft tissue sarcomas (STS): A planned analysis of the ISG-STS-1001 randomized trial.

11572 Background: The characteristics of immune contexture and its prognostic and predictive value in STS is left to be understood. This planned analysis of the ISG-STS-1001 trial, which compared neoadjuvant anthracycline + ifosfamide (AI) vs a histology-tailored (HT) chemotherapy (ChT), was aimed at characterizing the immune contexture after neoadjuvant ChT and investigating any association with the risk of recurrence. Methods: Patients registered in the ISG-STS-1001 study (ID: NCT01710176) were included if they had tumor tissue available for Tissue MicroArray (TMA), which was performed in the area of the surgical specimen with the highest lymphocyte infiltrate. The following markers were analyzed with IHC and measured quantitatively: CD3, CD8, PD1, GranzymeB, Foxp3, CD20, CD163, and PDL1. The T-Distributed Stochastic Neighboring Entities (t-SNE) analysis was used to account for the co-expression of IHC markers in each tumor. The prognostic value of each marker for disease-free survival (DFS) was assessed. Results: This analysis was conducted in 256 of 435 study patients. AI and HT neoadjuvant ChT did not result in any different distribution of immune contexture. Conversely, differences were observed between ‘complex’ karyotype STS (ck-STS: LMS, MPNST, UPS, MFS, pleomorphic liposarcoma, and pleomorphic rhabdomyosarcoma) and ‘simple’ karyotype STS (sk-STS: MLPS and SS). Ck-STS were enriched in both CD3+ and CD8+ cells compared to sk-STS. These cells displayed an heterogeneous distribution and were dispersed inside the tumor nest, keeping direct contact with sarcoma cells. Ck-STS also displayed an enrichment in Granzyme B+, and CD163+ cells. PDL1+ cells were occasionally identified and were more frequent in ck-STS, suggesting an immune-related expression. Most STS were negative for CD20+ cells, however, when present these cells were highly represented and organized in tertiary lymphoid-like structure. The t-SNE generated plot clustered tumors, the ‘cold’ mainly including sk-STS and the ‘hot’ mainly composed by ck-STS. In the ‘hot’ group, a cluster of tumors displayed an immune infiltrate enriched with a high number of CD3, CD8, GranzymeB, PD-1, and PDL-1+ cells. When the prognostic value of the immune markers was investigated, the presence of CD20+ cells was the only independent prognostic factor for DFS (HR=0.68, 95%CI 0.52-0.91) in a histology-stratified estimate adjusting for tumor size in cm (HR=1.07, 95%CI 1.03-1.12) and patient age (HR=1.0, 95%CI 0.97-1.02). Conclusions: Immune contexture differed across sarcoma histologies after neoadjuvant ChT, rather than across the two study arms, with ck-STS being marked by a rich immune contexture. While a CD20+ infiltrate was found to be an independent prognostic factor for a better outcome, further analyses are in progress on the prognosis of patients with the richest immune contexture. Clinical trial information: NCT01710176.

Head and neck rhabdomyosarcoma with TFCP2 fusions and ALK overexpression: a clinicopathological and molecular analysis of 11 cases.

Primary intraosseous rhabdomyosarcoma (RMS) is a rare entity defined by EWSR1/FUS-TFCP2 or, less commonly, MEIS1-NCOA2 fusions. The lesions often show a hybrid spindle and epithelioid phenotype, frequently coexpress myogenic markers, ALK, and cytokeratin, and show a striking propensity for the pelvic and craniofacial bones. The aim of this study was to investigate the clinicopathological and molecular features of 11 head and neck RMSs (HNRMSs) characterised by the genetic alterations described in intraosseous RMS. The molecular abnormalities were analysed with fluorescence in-situ hybridisation and/or targeted RNA/DNA sequencing. Seven cases had FUS-TFCP2 fusions, four had EWSR1-TFCP2 fusions, and none had MEIS1-NCOA2 fusions. All except one case were intraosseous, affecting the mandible (n=4), maxilla (n=3), and skull (n=3). One case occurred in the superficial soft tissue of the neck. The median age was 29years (range, 16-74years), with an equal sex distribution. All tumours showed mixed epithelioid and spindle morphology. Immunohistochemical coexpression of desmin, myogenin, MyoD1, ALK, and cytokeratin was seen in most cases. An intragenic ALK deletion was seen in 43% of cases. Regional and distant spread were seen in three and four patients, respectively. Two patients died of their disease. We herein present the largest series of HNRMSs with TFCP2 fusions to date. The findings show a strong predilection for the skeleton in young adults, although we also report an extraosseous case. The tumours are characterised by a distinctive spindle and epithelioid phenotype and a peculiar immunoprofile, with coexpression of myogenic markers, epithelial markers, and ALK. They are associated with a poor prognosis, including regional or distant spread and disease-related death.