Spread Through Air Spaces in Stage I to III Resected Lung Adenocarcinomas: Should the Presence of Spread Through Air Spaces Lead to an Upstaging?

In recent years, the concept of spread through air spaces (STAS) has been discussed as an adverse prognostic factor for lung cancer. The aim of our study is to clarify the prognostic role of STAS in relation to the main recognized prognostic factors in a retrospective cohort of 330 European patients who underwent stages I to III lung adenocarcinoma resection. On univariate analysis, the presence of STAS was related to progression-free survival (PFS; hazard ratio [HR]: 1.48; 95% CI: 1.02-2.19; P = 0.038) and overall survival (OS; HR: 1.61; 95% CI: 1.03-2.52; P = 0.50). On multivariate analysis, STAS was related to PFS (HR: 1.51; 95% CI: 1.00-2.17; P = 0.050) and to OS (HR: 1.67; 95% CI: 1.00-2.81; P = 0.050). We showed that the presence of STAS was associated with lower PFS, equivalent to the next pathologic T stage, especially the median PFS of T3 stages without STAS was at 62.8 months while the median PFS of T3 stages with STAS was at 15.7 months, closer to the median PFS of 17.4 months in T4 stages. To conclude, STAS is an independent prognostic factor of PFS in this European cohort and is close to significance for OS. We suggest that the presence of STAS might lead to an upstaging of lung adenocarcinoma.

Immunohistochemical and Molecular Characteristics of Anogenital Papillary Hidradenomas.

BACKGROUND: Papillary hidradenomas (PHs) of the anogenital region are uncommon tumors whose immunohistochemical and molecular profile have been infrequently studied. MATERIAL AND METHODS: We studied 15 PHs by next-generation sequencing and 10 immunohistochemical markers (PAX8, GATA3, HER2, MSH6, PMS2, estrogen, progesterone and androgen receptors, CK14, and NKX3.1). RESULTS: All cases expressed GATA3, whereas none expressed PAX8, and rare tumor cells were NKX3.1-positive. Almost all cases expressed estrogen receptors (ER), progesteron receptors (PR), and androgen receptors (AR). CK14 was expressed by myoepithelial cells, whereas only rarely by the epithelial tumor cells. HER2 showed no significant expression. Immunohistochemical expression for the mismatch repair proteins showed persistence in all cases. Molecular analysis often showed PIK3CA mutations, as well as KRAS , SMO , and MAP2K1 mutations. CONCLUSION: Anogenital PHs frequently harbor PIK3CA mutations and show a PAX8-, GATA3/ER/PR/AR + immunohistochemical profile.

Uterine inflammatory myofibroblastic tumor with THBS1-INSR fusion.

Uterine inflammatory myofibroblastic tumors (IMTs) are rare and, as in other localisations, they are associated with ALK rearrangements and ALK immunohistochemical expression. They are more frequently found during pregnancy, and in this context, they show different characteristics compared to other uterine IMTs. Here, we report the case of a uterine IMT discovered during delivery, and being associated with a previously unreported THBS1-INSR fusion.

Peripheral node addressin, a ligand for L-selectin is found in tumor cells and in high endothelial venules in endometrial cancer.

BACKGROUND: High endothelial venules (HEVs) are vessels specialized in the transport of lymphocytes shown to be implicated in various forms of cancer. They express peripheral node addressin (specifically recognized by the MECA-79 antibody). MECA-79 is also implicated in pregnancy through its expression by epithelial cells of the endometrium. However, the expression of MECA-79 by endothelial or epithelial (cancer) cells has never been studied in endometrial cancer. MATERIAL AND METHODS: In this retrospective study, we investigated the immunohistochemical expression of MECA-79 in 40 endometrioid adenocarcinoma hysterectomy specimens and compared it with its expression in 30 non-cancer hysterectomies. RESULTS: HEVs were found in 22% of tumor specimens and in none of the non-cancer hysterectomies (p = 0.005) and were positively associated with higher grade tumors (p = 0.04). MECA-70 was expressed in tumor cells of 70% of carcinomas and in epithelial cells of 46.6% of normal endometria (p = 0.04). It was inversely associated with parametrial invasion (p = 0.03) and larger tumors (statistical trend of p = 0.07). MECA-79 expression was not associated with overall or progression-free survival. CONCLUSION: MECA-79 is found in HEVs and tumor cells in endometrial endometrioid adenocarcinoma.

WHO grading system for invasive pulmonary lung adenocarcinoma reveals distinct molecular signature: An analysis from the cancer genome atlas database.

Lung adenocarcinoma grading has gained interest in the past years. Recently a three-tier tumor grading was proposed showing that it is related to patients' prognosis. Nevertheless, the underlying molecular basis of this morphological grading remains partly unknown. The aim of our work is to take advantage of The Cancer Genome Atlas lung adenocarcinoma (TCGA_LUAD) cohort to describe the molecular data associated to tumor grading. We performed a study on publicly available data of the TCGA database first by assessing a tumor grade on downloadable tumor slides. Secondly we analyzed the molecular features of each tumor grade group. Our work was performed on a study group of 449 patients. We show that aneuploidy score was significantly different between grade 2 and grade 3 groups with different chromosomal imbalance (p < 0.001). SCGB1A1 mRNA expression was higher in grade 2 (p = 0.0179) whereas NUP155, CHFR, POLQ and CDC7 have a higher expression in grade 3 (p = 0.0189, 0.0427, 0.0427 and 0.427 respectively). GZMB and KRT80 have a higher methylation of DNA in grade 2 (p = 0.0201 and 0.0359 respectively). MT1G, CLEC12B and NDUFA7 have a higher methylation of DNA in grade 3 (p < 0.001, 0.0246 and 0.0359 respectively). We showed that the number of activated pathways is different between grade 2 and grade 3 patients (p = 0.004). We showed that differentially expressed genes by mRNA analysis and DNA methylation analysis involve several genes implied in chemoresistance. This could suggest that grade 3 lung adenocarcinoma might be more resistant to chemotherapy.

Molecular Analysis of an Abdominal Wall Cesarean Section Endometrioid Carcinoma.

Malignant transformation of endometriosis is rare, and most cases concern the ovaries, while extraovarian cases are mostly found in the rectovaginal septum. Incisional adenocarcinoma is extremely rare, with only few cases reported in the literature, while their molecular profile remains unknown. Thus, we report on an abdominal wall cesarean section scar endometrioid adenocarcinoma studied by next-generation sequencing and microsatellite instability analysis.

Histopathological and molecular profiling of lung adenocarcinoma skin metastases reveals specific features.

AIMS: Little is known regarding the histopathological and molecular features of lung adenocarcinoma skin metastases. Our study is the largest, to our knowledge, to comprehensively explore these to date. METHODS AND RESULTS: We performed a retrospective cohort study analysing 42 lung adenocarcinoma skin metastasis samples obtained from a database of 2659 lung adenocarcinomas collected between 2010 and 2020. EGFR exon 19 deletion was detected in one patient and KRAS mutations were detected in 12 (33.3%) patients. The programmed cell death ligand 1 (PD-L1) tumour proportion score was <1% in 27 patients, ≥1% and <50% in eight patients, ≥50% in six patients and not assessable in one patient. We showed that the predominant histopathological subtype is different from that at other metastatic sites (P = 0.024). Thyroid transcription factor I (TTF-1) was more often negative in skin metastases compared to other sites (P < 0.001). The EGFR mutation rate tended to be lower for skin metastases compared to other sites (P = 0.079). Skin metastases were associated with a high rate of PD-L1-negative cases (P = 0.022). CONCLUSION: Our work shows that the skin metastases of lung adenocarcinoma have a specific histopathological profile.

Senescence, immune microenvironment, and vascularization in cardiac myxomas.

AIMS: Cardiac myxomas are rare tumors of incompletely elucidated pathogenesis. The aim of this study is to explore the possible presence of a senescence phenotype in cardiac myxomas, associated with an inflammatory and vasculogenic tumor microenvironment. METHODS AND RESULTS: This is a retrospective study of 29 cardiac myxomas with immunohistochemical detection of various inflammatory, vascular, and senescence markers. We show that all myxomas contain tumor cells in senescence overexpressing p16, and a fraction of senescent endothelial cells. Macrophages are the principal inflammatory cell population, followed by cytotoxic T cells, with fewer plasma cells, mastocytes, and B lymphocytes. These populations are found in different intratumoral localizations. Larger tumor volume is associated with a lower percentage of myxoid matrix, higher cellularity, higher macrophage, and lower number of mast cells as well as higher PD-L1 expression by inflammatory cells. Higher vascular density is associated with higher percentage of B cells, a lower number of macrophages and higher number of mastocytes, and lower PD-L1 expression by inflammatory cells. Tumors with higher vascular density and higher cellularity show higher amounts of p16 senescent endothelial cells. CONCLUSIONS: Myxoma tumor cells are in senescence and reside inside a tumor microenvironment with a distinct inflammatory profile rich in macrophages and cytotoxic T cells, and a rich vasculature, probably attributed to a senescence-associated secretory phenotype.

Heterogeneity of PD-L1 expression in lung adenocarcinoma metastasis is related to histopathological subtypes.

OBJECTIVES: The heterogeneity of PD-L1 expression and its relationship with histopathological subtype has recently been shown on primary tumor but has not been evaluated on metastases. The aim of our work is to analyze PD-L1 expression within each histopathological pattern on resected metastases. MATERIAL AND METHODS: 136 patients were included in this retrospective study. Immunohistochemistry was performed with 22C3 laboratory-developed test. The Tumor Proportion Score was evaluated on each subtype. RESULTS: The most frequent major histopathological subtype was solid (n = 69, 50.7 %), followed by acinar (n = 37, 27.2 %), micropapillary (n = 14, 10.3 %) and papillary (n = 10, 7.3 %). Mean percentage of PD-L1 expression for each subtype was at 28+/-4.8 % for solid subtype, 5.3+/-1.9 % for acinar subtype, 5+/-1.9 % for papillary subtype and 23.6+/-4.1 % for micropapillary subtype. Mean percentage of PD-L1 expression was different between solid pattern and acinar pattern (p < 0.001), solid pattern and papillary pattern (p = 0.007), micropapillary pattern and acinar pattern (p < 0.001) and micropapillary pattern and papillary pattern (p = 0.015). CONCLUSION: To conclude, we have showed firstly that several patterns are present in metastases of lung adenocarcinoma, secondly that the evaluation of patterns and PD-L1 stain on different patterns is reproducible, thirdly that pattern heterogeneity is related to PD-L1 staining, fourthly that in metastatic lung adenocarcinoma with at least two patterns, solid and micropapillary subtypes have higher levels of PD-L staining, fifthly that PD-L1 heterogeneity between different patterns is not a rare event. These results might explain discrepancies of PD-L1 results between biopsies and surgical samples and the fact that some patients might respond to checkpoint inhibitors even though PD-L1 expression is low or absent.

Molecular analysis of a uterine broad ligament leiomyoma in a patient with CLOVES syndrome.

Overgrowth syndromes are characterized by a global or regional excess growth of various tissue types, especially of mesenchymal nature. The CLOVES (Congenital Lipomatous asymmetric Overgrowth of the trunk with lymphatic, capillary, venous, and combined-type Vascular malformations, Epidermal naevi, Scoliosis/Skeletal and spinal anomalies) syndrome is characterized by an asymmetric growth excess associated with PIK3CA mutations, found in mosaic, affecting the lesional, but not the normal tissues. Herein, we report the case of a patient affected by CLOVES syndrome, harboring a 13 cm leiomyoma of the uterine broad ligament. The leiomyoma tissue was examined by next-generation sequencing showing the absence of related mutations.

Brain metastasis PD-L1 and CD8 expression is dependent on primary tumor type and its PD-L1 and CD8 status.

BACKGROUND: Brain metastases (Bmets) are frequent; however, limited data exist on the efficacy of immunotherapy in these lesions. The aims of the study were to analyze the immunohistochemical expressions of programmed death ligand 1 (PD-L1) and CD8 in Bmets and to compare them with their expressions in paired primary tumors, as well as correlate the results with clinicopathological features. METHODS: This is a retrospective study of 233 patients with Bmets and 111 paired primaries. Clinical, histological, and molecular data were recorded and compared with the immunohistochemical results of PD-L1 and CD8 expressions. The statistical analysis included χ2 test, Cramer's V test, factorial analyses of variance, simple regression analysis, and Kaplan-Meier analysis with log-rank product limit estimation. RESULTS: PD-L1 expression was found in 23.6% of Bmets and in 29.0% of primary tumors with concordant expression between them in 75.5% of cases. Bmets PD-L1 expression was associated with primary tumor PD-L1 expression and the primary tumor type. Significant CD8 peritumoral expression was found in 68.6% of Bmets and in 87.7% of primary tumors. CD8 expression was concordant between primary and metastatic tumors in 73.3% of cases. Bmets CD8 expression was associated with primary tumor CD8 expression and primary tumor type. PD-L1 expression was associated with CD8 expression in both primary and metastatic tumors. The concordance between primary and metastatic tumor PD-L1 expression was independent of all factors studied. The concordance between primary and metastatic CD8 expressions was marginally associated to the time of Bmets development. No prognostic role for PD-L1 and CD8 expression in Bmets was found. CONCLUSION: PD-L1 and CD8 Bmets expressions are associated with the primary tumor type and its PD-L1 and CD8 expressions. No factor predicts the discordance for PD-L1 expression, while time to Bmets development is associated with CD8 expression discordance.

FAK Deficiency in Bone Marrow Stromal Cells Alters Their Homeostasis and Drives Abnormal Proliferation and Differentiation of Haematopoietic Stem Cells.

Emerging evidence indicates that in myelodysplastic syndromes (MDS), the bone marrow (BM) microenvironment may also contribute to the ineffective, malignant haematopoiesis in addition to the intrinsic abnormalities of haematopoietic stem precursor cells (HSPCs). The BM microenvironment influences malignant haematopoiesis through indirect mechanisms, but the processes by which the BM microenvironment directly contributes to MDS initiation and progression have not yet been elucidated. Our previous data showed that BM-derived stromal cells (BMSCs) from MDS patients have an abnormal expression of focal adhesion kinase (FAK). In this study, we characterise the morpho-phenotypic features and the functional alterations of BMSCs from MDS patients and in FAK knock-downed HS-5 cells. The decreased expression of FAK or its phosphorylated form in BMSCs from low-risk (LR) MDS directly correlates with BMSCs' functional deficiency and is associated with a reduced level of haemoglobin. The downregulation of FAK in HS-5 cells alters their morphology, proliferation, and differentiation capabilities and impairs the expression of several adhesion molecules. In addition, we examine the CD34+ healthy donor (HD)-derived HSPCs' properties when co-cultured with FAK-deficient BMSCs. Both abnormal proliferation and the impaired erythroid differentiation capacity of HD-HSPCs were observed. Together, these results demonstrate that stromal adhesion mechanisms mediated by FAK are crucial for regulating HSPCs' homeostasis.

Parathyroid Hormone Remodels Bone Transitional Vessels and the Leptin Receptor-Positive Pericyte Network in Mice.

Intermittent parathyroid hormone (iPTH) is anti-osteoporotic and affects bone vessels. Transitional capillaries close to the bone surface, which express both endomucin (Edm) and CD31, bear leptin receptor-expressing (LepR) perivascular cells that may differentiate into osteoblasts. Increased numbers of type H endothelial cells (THEC; ie, Edmhi /CD31hi cells assessed by flow cytometry, FACS) are associated with higher bone formation in young mice. We hypothesized that iPTH administration impacts transitional vessels by expanding THECs. Four-month-old C57/Bl6J female mice were injected with PTH 1-84 (100 µg/kg/d) or saline (CT) for 7 or 14 days. We quantified LepR+ , CD31+ , Edm+ cells and THECs by FACS in hindlimb bone marrow, and Edm/LepR double immunolabelings on tibia cryosections. Additionally, we analyzed bone mRNA expression of 87 angiogenesis-related genes in mice treated with either intermittent or continuous PTH (iPTH/cPTH) or saline (CT) for 7, 14, and 28 days. iPTH dramatically decreased the percentage of THECs by 78% and 90% at days 7 and 14, respectively, and of LepR+ cells at day 14 (-46%) versus CT. Immunolabeling quantification showed that the intracortical Edm+ -vessel density increased at day 14 under iPTH. In the bone marrow, perivascular LepR+ cells, connected to each other via a dendrite network, were sparser under iPTH at day 14 (-58%) versus CT. iPTH decreased LepR+ cell coverage of transitional vessels only (-51%), whereas the number of LepR+ cells not attached to vessels increased in the endocortical area only (+ 49%). Transcriptomic analyses showed that iPTH consistently upregulated PEDF, Collagen-18α1, and TIMP-1 mRNA expression compared with CT and cPTH. Finally, iPTH increased immunolabeling of endostatin, a Collagen-18 domain that can be cleaved and become antiangiogenic, in both endocortical (79%) and peritrabecular transitional microvessels at day 14. Our results show that iPTH specifically remodels transitional vessels and suggest that it promotes LepR+ cell mobilization from these vessels close to the bone surface. © 2019 American Society for Bone and Mineral Research.

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation.

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.

Evaluation by Flow Cytometry of Mature Monocyte Subpopulations for the Diagnosis and Follow-Up of Chronic Myelomonocytic Leukemia.

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative neoplasm, characterized by persistent monocytosis and dysplasia in at least one myeloid cell lineage. This persistent monocytosis should be distinguished from the reactive monocytosis which is sometimes observed in a context of infections or solid tumors. In 2015, Selimoglu-Buet et al. observed an increased percentage of classical monocytes (CD14+/CD16- >94%) in the peripheral blood (PB) of CMML patients. In this study, using multiparametric flow cytometry (MFC), we assessed the monocytic distribution in PB samples and in bone marrow aspirates from 63 patients with monocytosis or CMML suspicion, and in seven follow-up blood samples from CMML patients treated with hypomethylating agents (HMA). A control group of 12 healthy age-matched donors was evaluated in parallel in order to validate the analysis template. The CMML diagnosis was established in 15 cases in correlation with other clinical manifestations and biological tests. The MFC test for the evaluation of the repartition of monocyte subsets, as previously described by Selimoglu-Buet et al. showed a specificity of 97% in blood and 100% in marrow samples. Additional information regarding the expression of intermediate MO2 monocytes percentage improved the specificity to 100% in blood samples allowing the screening of abnormal monocytosis. The indicative thresholds of CMML monocytosis were different in PB compared to BM samples (classical monocytes >95% for PB and >93% for BM). A decrease of monocyte levels in PB and BM, along with a normalization of monocytes distribution, was observed after treatment in 4/7 CMML patients with favorable evolution. No significant changes were observed in 3/7 patients who did not respond to HMA therapy and also presented unfavorable molecular prognostic factors at diagnosis (ASXL1, TET2, and IDH2 mutations). Considering its simplicity and robustness, the monocyte subsets evaluation by MFC provides relevant information for CMML diagnosis.

Epigallocatechin-3-O-gallate alleviates the malignant phenotype in A-431 epidermoid and SK-BR-3 breast cancer cell lines.

In this study, we evaluated the effects of epigallocatechin-3-O-gallate (EGCG) in two cancer cell lines, A-431 overexpressing ErbB1 and SK-BR-3, overexpressing ErbB2. EGCG treatment showed dose-dependent collapse of mitochondrial membrane potential (Δψm), increase in reactive oxygen species (ROS) production, changes in nuclear morphology and reduced viability. Flow cytometry data indicated that EGCG partially decreases the phosphorylation of several proteins involved in cell proliferation and survival: pErbB1(Y1173, Y1068), pAkt(S473) and pERK(Y204). EGCG affected the clonogenic growth in both cell lines with an EC50 of 2.5 and 5.4 µM for A-431 and SK-BR-3, respectively. Wound scratch assay demonstrated that EGCG inhibited the healing in dose-dependent manner and the effect was correlated with partial reduction in phosphorylation of pFAK(S910). Our data suggest that EGCG administration might reduce the unfavourable traits, particularly associated with ErbB1/EGFR overexpression.