The Premetastatic Lymph Node Niche in Gynecologic Cancer.

It has been suggested that a primary tumor can "prepare" the draining of lymph nodes to "better accommodate" future metastatic cells, thus implying the presence of a premetastatic lymph node niche. However, this phenomenon remains unclear in gynecological cancers. The aim of this study was to evaluate lymph-node draining in gynecological cancers for premetastatic niche factors, such as myeloid-derived suppressor cells (MDSCs), immunosuppressive macrophages, cytotoxic T cells, immuno-modulatory molecules, and factors of the extracellular matrix. This is a monocentric retrospective study of patients who underwent lymph-node excision during their gynecological-cancer treatment. In all, 63 non-metastatic pelvic or inguinal lymph nodes, 25 non-metastatic para-aortic lymph nodes, 13 metastatic lymph nodes, and 21 non-cancer-associated lymph nodes (normal controls) were compared for the immunohistochemical presence of CD8 cytotoxic T cells, CD163 M2 macrophages, S100A8/A9 MDSCs, PD-L1+ immune cells, and tenascin-C, which is a matrix remodeling factor. PD-L1-positive immune cells were significantly higher in the control group, in comparison to the regional and distant cancer-draining lymph nodes. Tenascin-C was higher in metastatic lymph nodes than in both non-metastatic nodes and control lymph nodes. Vulvar cancer-draining lymph nodes showed higher PD-L1 values than endometrial cancer and cervical cancer-draining lymph nodes. Endometrial cancer-draining nodes had higher CD163 values and lower CD8 values, compared to vulvar cancer-draining nodes. Regarding regional draining nodes in low- and high-grade endometrial tumors, the former showed lower S100A8/A9 and CD163 values. Gynecological cancer-draining lymph nodes are generally immunocompetent, but vulvar cancer draining nodes, as well as high-grade endometrial cancer draining nodes, are more susceptible to harboring premetastatic niche factors.

Crystal-Storing Histiocytosis: The Iceberg of More Serious Conditions.

Crystal-storing histiocytosis is a rare condition that is histologically characterized by intracellular cytoplasmic crystalline inclusions. It usually presents monoclonal immunoglobulins that deposit within histiocytes, which accumulate and affect different organs of the human body and are commonly associated with lymphoproliferative conditions, especially those with plasmacytic differentiation. The prognosis of this condition is variable and related to the underlying clinical disease. In this review article, we aim to describe and discuss the clinical and pathological characteristics of crystal-storing histiocytosis based on the available literature and to provide a thorough differential diagnosis.

PD-L1 expression in head and neck cancer tissue specimens decreases with time.

BACKGROUND: PD-L1 immunohistochemical expression is used as an important theranostic marker in various malignancies, including head and neck squamous cell carcinoma (HNSCC) where the combined positive score (CPS) guides treatment decisions. Despite indirect evidence that there is loss of antigenicity for archived tissues, there is no direct comparison between PD-L1 expression of the same tissue upon arrival and after its storage. MATERIAL AND METHODS: We compared the immunohistochemical expression of PD-L1 (22C3) in 106 HNSCC upon their arrival and after their storage (interval ranging from 20 to 48 months, mean 30.8 months). The evaluation was performed by two different pathologists' groups. RESULTS: We found a statistically significant decrease in the PD-L1 tumor proportional score (TPS), immune cells expression (IC) and CPS between the initial and the newly stained slides. CONCLUSION: This is the first study comparing PD-L1 expression between a tissue and "himself" later in time, highlighting an important decrease in expression by tumor and immune cells, and suggesting that an immediate rather than a retrospective assay of PD-L1 expression should be preferable in the routine practice. DATA AVAILABILITY: Data are available upon reasonable request.

STAT6 and phosphorylated STAT6 are differentially expressed in lymphomas.

BACKGROUND: The STAT6 pathway is implicated in the pathogenesis of various lymphomas; however, its immunohistochemical expression has not been fully investigated. Thus, the aim of this study was to investigate the immunohistochemical expression of the two forms of STAT6, phosphorylated or not, in a series of systemic lymphomas. MATERIALS AND METHODS: Immunohistochemical expression of two antibodies, STAT6 (clone YE361) and pSTAT6 (clone Y641), which recognise the phosphorylated form of the molecule was studied in 60 lymphomas. RESULTS: STAT6YE361 expression was cytoplasmic, with 23.3% of the cases showing high expression. pSTAT6Y641 expression was mostly nuclear and found in 45% of the cases. pSTAT6Y641 nuclear expression was associated with the lymphoma type (p < 0.0001), as it was seen mostly in follicular, Hodgkin and angioimmunoblastic T cell lymphomas. STAT6YE361 cytoplasmic expression was also associated with lymphoma type (p = 0.001), as it was mostly found in diffuse large B cell and marginal B cell lymphomas. No association with PD-L1 expression, other clinicopathological data or prognosis was found. CONCLUSION: The two STAT6 clones are differentially expressed between lymphoma types.

Primary central nervous system lymphomas express immunohistochemical factors of autophagy.

Primary central nervous system lymphoma (PCNSL) is an aggressive and rare disease. Autophagy is a catabolic mechanism boosting various tumors, including lymphomas; its inhibition is thus a promising therapeutic target. Its presence has never been studied in PCNSLs. We conducted a retrospective immunohistochemical study of 25 PCNSLs for LC3B, p62, and M6PR, comparing it with clinicopathological characteristics. Fourteen (56%) and eleven (44%) PCNSLs were of low and high LC3B expression, respectively. p62 expression was present in most tumors (n = 21, 84%). M6PR was present in all tumors, with 14 (56%) and 11 (44%) cases being of low and high M6PR expression, respectively. LC3B expression was correlated with the performance status (PS) (p = 0.04). No association was found with other clinical parameters, such as deep structure invasion, multiple lesions, complete response, and recurrence after response. p62 showed a strong positive association with MUM1 expression (p = 0.0005). M6PR expression showed a positive correlation (p = 0.04) with PD-L1 expression. No association was found with p53, Ki67, CD8, BCL2, BCL6, or double MYC/BLC2 co-expressors. No association of LC3B, p62, and M6PR expression with survival was found. Our findings provide evidence for the possible presence of autophagic markers in PCNSLs and, thus, for possible treatment targets.

Autophagy and immune microenvironment in craniopharyngioma and ameloblastoma.

BACKGROUND: Craniopharyngiomas and ameloblastomas show remarkable histologic and molecular similarities. The immune microenvironment of craniopharyngiomas has been recently studied showing interesting findings, while its composition in ameloblastomas is unknown. Similarly, some evidence of autophagic activity, a process of cellular constituents' degradation has been found in ameloblastomas, but no studies exist in craniopharyngiomas. Thus, the aim of the study is to compare factors of the immune microenvironment and the autophagic apparatus between these two tumor types. METHODS: 26 craniopharyngiomas and 14 ameloblastomas were immunohistochemically studied for PD-L1, CD8, CD20, S100, CD163, MECA-79, LC3B and p62. RESULTS: Craniopharyngiomas showed higher LC3B tumor cell expression, higher CD8+ T cells and higher CD163+ macrophages in comparison to ameloblastomas. LC3B tumor cell expression was associated with overall survival in craniopharyngioma patients and p62 nuclear expression was associated with overall survival in ameloblastoma patients. CONCLUSION: This is the first study showing the presence of autophagic markers in craniopharyngiomas and describing the immune microenvironment of ameloblastomas.

Autophagy in cardiac myxoma: An important puzzle piece in understanding its inflammatory environment.

BACKGROUND: Cardiac myxomas are rare, predominantly sporadic tumors that can cause heart failure and systematic inflammatory symptoms, and increase the risk of emboli. Their pathophysiology remains poorly understood, but intra-tumoral inflammation and senescence seem to be implicated in it. One of the principal cellular mechanisms implicated in tumor progression is autophagy, largely unknown in myxomas. Thus, our study aimed to investigate the presence of autophagic markers in myxomas and to correlate it with their immune microenvironment. METHODS: Twenty-five cardiac myxomas were studied for the autophagic markers LC3B and p62/sequestosome 1 and were compared with markers of the immune microenvironment. RESULTS: Most myxomas showed expression of both autophagic markers. We found a positive correlation between LC3B and PD-L1, as well as CD163, and a negative correlation between LC3B and CD8, CD20, CD138, and CD117 infiltration. CONCLUSION: Our data not only confirm the presence of autophagic markers within cardiac myxomas but also suggest a possible association with their immune microenvironment.

Expression of STAT6 and Phosphorylated STAT6 in Primary Central Nervous System Lymphomas.

The signal transducer and activator of transcription 6 (STAT6) is implicated in the pathogenesis of some lymphomas including primary central nervous system lymphomas (PCNSLs). The aim of this study was to investigate STAT6 expression and clinicopathologic features in 25 PCNSLs using immunohistochemistry with 2 different anti-STAT6 antibodies. One (YE361) recognizes the C-terminus domain of the STAT6 protein and the other (Y641) recognizes the phosphorylated form of the protein. The phosphorylated STAT6 form was not expressed in any of the cases studied whereas the YE361 STAT6 showed only cytoplasmic expression in 14 (56%) cases. This expression did not correlate with age, prognostic score, multiplicity, invasion of deep structures, response to treatment, disease recurrence, overall survival, or BCL6, BCL2, PD-L1, and CD8 expression. A STAT6 expression score showed a trend for correlating with clinical performance status. It also showed a positive correlation with MYC expression. Thus, the phosphorylated form of STAT6 was not found in the current series, while the YE361 STAT6 showed only cytoplasmic expression and was associated with expression of MYC.

Dual laryngeal reinnervation in bilateral vocal fold paralysis: anatomical pitfalls.

BACKGROUND: Bilateral laryngeal reinnervation can be a promising procedure for reanimation of laryngeal muscles, but currently not yet standardized. Besides patient conditions some intraoperative anatomical pitfalls need to be solved. METHODS: Twelve human head and neck specimens (24 sides) have been studied using microdissection and histological serial sections of the nerves. The surgical anatomy of the dual reinnervation procedure according to JP Marie was investigated notably the branching pattern of the phrenic nerve (PN), the Ansa cervicalis (AC) and the recurrent laryngeal nerve (RLN). RESULTS: Despite variations of the AC, a prominent inferior common trunk for sterno-hyoid and sterno-thyroid muscles can be used in more than 90% of the specimens. If the AC is missing because of previous surgery, the tiny nerve of the thyro-hyoid muscle can be used preferred. The PN display a double roots pattern from C3 to C4 cervical plexus in 50% of the specimens. A single root pattern can be found and an end-to-lateral neurorraphy can be used. Intra-laryngeal nerves pattern of the RLN display tiny collaterals which cannot be selected for abduction-adduction activity. Direct implantation of the Y-shape great auricular nerve within the posterior crico-arytenoid muscles can be a reliable method leading to challenging mechanical and functional conditions. CONCLUSION: Several anatomical pitfalls, including intra-operative choices and variants of the donor nerves, but also the challenging intra-laryngeal dissection of the inferior laryngeal nerve need to be solved. A successful laryngeal reinnervation still needs further studies for a simplified procedure.

The Immune Microenvironment of Chordomas: An Immunohistochemical Analysis.

Chordomas are rare sarcomas that are usually treated by surgery and/or radiotherapy since these are chemo-resistant tumors, but immunotherapy could be a possible option for chordoma patients. However, few reports investigating the composition of the chordoma immune microenvironment exist. We immunohistochemically studied 81 chordomas regarding their immune microenvironment factors and compared them with clinicopathological data. Macrophages and CD4 cells were the most prominent inflammatory cell populations, followed by CD8 T cells, while CD20 B cells and high endothelial venules (MECA-79+) were less frequently found. PD-L1 (22C3) expression by inflammatory cells was found in 21 (26%) tumors and was associated with a larger tumor size. None of the cases showed the expression of PD-L1 by tumor cells. Survival analysis showed that younger patients had a better overall survival. Considering the immunohistochemical factors studied, higher CD8, the presence of PD-L1+ immune cells, and higher vascular density were adverse prognostic factors, but in multivariate analysis, only PD-L1+ immune cells retained prognostic significance. To conclude, chordoma tumor cells do not express PD-L1, but PD-L1+ immune cells seem to play a negative prognostic role, supporting the need for further studies in this field and the possible beneficial role of immunotherapy in these patients.

Proteomic Imaging of Vestibular Schwannomas and Normal Nerves. Histopathologic Correlations.

OBJECTIVES: Proteomic analysis of vestibular schwannoma (VS), non-vestibular schwannoma (NVS), and normal nerve (NN) using mass spectrometry and imaging of matrix assisted laser desorption ionization-time of flight (MALDI-TOF). METHODS: Retrospective, qualitative, and descriptive study on VS, NVS, and NN. Samples were provided by our Tumor Bank. They were analyzed histologically then sprayed by acid matrix. The laser beam of MALDI performed desorption-ionization of the sample. A mass spectrogram (MS) was drawn depending on time of flight of ionized peptides, and MALDI-imaging was obtained which is a summation color spectrum depending on sample's peptide content. The slice was reexamined histologically and results compared with MALDI-imaging. RESULTS: Fifty schwannomas were sampled, of which 27 exploitable: 22 VS (17 Antoni type A and five type B) and five NVS (all Antoni type B). Eleven NN were analyzed. Among the 22 VS, near-total correlation between MALDI-imaging and pathology was found in two cases (9.1%), partial correlation in four (18.2%), and no correlation in 16 (72.7%); correlations were more frequent in VS of the Antoni type B. MS showed a peptide spike at 2,000 m/z in 7 (31.8%) and 5,000 m/z in 21 (95.5%). Among the five NVS, near-total correlation was found in three cases (60%), partial correlation in one (20%), and no correlation in one (20%). MS showed a peptide spike at 2,000 m/z in two (40%) and 5,000 m/z in all (100%). Among the 11 NN, near-total correlation was found in nine cases (81.8%), partial correlation in one (9.1%), and no correlation in one (9.1%). MS showed no peptide spike at 2,000 or 5,000 m/z. Behind homogeneous areas on histology, there was great heterogeneity on MALDI-imaging and MS, regarding VS and NVS, but not NN. CONCLUSIONS: There was a lack of correlation between MALDI-imaging and pathology in VS (except Antoni type B) as compared with NVS and NN. The lack of correlation in VS of the type A as compared with type B VS and NVS could be attributed to the overexpression of degeneration-associated proteins/peptides in VS of the type B as well as NVS that are better correlated with histologic findings. The two peptide spikes detected in schwannoma and not in NN opens up the prospect of tumor biomarkers identifiable by sequencing. The proteomic polymorphism found in VS and NVS was absent on histology which is a new morphologic characteristic of schwannoma. Further studies should be performed in the future to confirm the benefit and usefulness of the MALDI in the analysis of VS and NVS.

Autophagic Markers in Chordomas: Immunohistochemical Analysis and Comparison with the Immune Microenvironment of Chordoma Tissues.

Chordomas are notably resistant to chemotherapy. One of the cytoprotective mechanisms implicated in chemoresistance is autophagy. There are indirect data that autophagy could be implicated in chordomas, but its presence has not been studied in chordoma tissues. Sixty-one (61) chordomas were immunohistochemically studied for autophagic markers and their expression was compared with the expression in notochords, clinicopathological data, as well as the tumor immune microenvironment. All chordomas strongly and diffusely expressed cytoplasmic p62 (sequestosome 1, SQSTM1/p62), whereas 16 (26.2%) tumors also showed nuclear p62 expression. LC3B (Microtubule-associated protein 1A/1B-light chain 3B) tumor cell expression was found in 44 (72.1%) tumors. Autophagy-related 16­like 1 (ATG16L1) was also expressed by most tumors. All tumors expressed mannose-6-phosphate/insulin-like growth factor 2 receptor (M6PR/IGF2R). LC3B tumor cell expression was negatively associated with tumor size, while no other parameters, such as age, sex, localization, or survival, were associated with the immunohistochemical factors studied. LC3B immune cell expression showed a significant positive association with programmed death-ligand 1 (PD-L1)+ immune cells and with a higher vascular density. ATG16L1 expression was also positively associated with higher vascular density. Notochords (n = 5) showed different immunostaining with a very weak LC3B and M6PR expression, and no p62 expression. In contrast to normal notochords, autophagic factors such as LC3B and ATG16L1 are often present in chordomas, associated with a strong and diffuse expression of p62, suggesting a blocked autophagic flow. Furthermore, PD-L1+ immune cells also express LC3B, suggesting the need for further investigations between autophagy and the immune microenvironment.

Immunohistochemical analysis of L1 cell adhesion molecule and high endothelial venules in breast cancer brain metastasis.

BACKGROUND: The vasculature is a crucial factor in tumor development. Vascular co-option achieved by the L1 cell adhesion molecule (L1CAM) and lymphocyte recruitment inside tumors by high endothelial venules (HEVs) are important prognostic factors in primary breast cancer. Their status in breast cancer brain metastasis is unknown. AIM OF THE STUDY: To explore the status of L1CAMs and HEVs in this tumor compartment. MATERIAL AND METHODS: Thirty resected breast cancer brain metastases were immunohistochemically studied for L1CAM and MECA-79, an HEV marker. Clinicopathological factors were recorded. RESULTS: Age at brain metastasis diagnosis ranged from 37 to 80 years (median 55). The time to brain metastasis development after primary tumor diagnosis ranged from 12 to 187 months (median 57). Median overall survival after brain metastasis diagnosis was 29 months. None of the tumors expressed the factors studied. CONCLUSION: L1CAM and high endothelial venules are not found in breast cancer brain metastasis.

STAT6: A review of a signaling pathway implicated in various diseases with a special emphasis in its usefulness in pathology.

Signal Transducer and Activator of Transcription 6 (STAT6), belonging to a family of seven similar members is primarily stimulated by interleukin(IL)-4 and IL-13, and acts as a T helper type 2 (Th2)-inducing factor. Thus, it is implicated in the pathophysiology of various allergic conditions, such as asthma, atopic dermatitis, eosinophilic esophagitis and food allergies, but also in tumor microenvironment regulation. Furthermore, certain forms of lymphomas, notably the Hodgkin lymphoma group, the primary mediastinal and primary central nervous system lymphoma, as well as some follicular and T cell lymphomas are associated with dysregulation of the STAT6 pathway. STAT6 immunohistochemical expression also serves as a surrogate marker in the diagnosis of solitary fibrous tumor, despite not directly responsible for the tumorigenic effect. These pathophysiological implications of the STAT6 pathway, its diagnostic or prognostic role in pathology, as well its immunohistochemical detection with different antibodies will be discussed in this review.

Patterns of brachyury expression in chordomas.

INTRODUCTION: Chordomas are rare malignant midline tumors, presumed to arise from notochordal remnants. This was further suggested by the discovery of the brachyury in chordomas pathogenesis. Its immunohistochemical expression has become the principal adjunct in the diagnosis of chordomas. However, studies about brachyury expression in chordomas are not fully comparable, mainly because they use different primary antibodies. Thus, the aim of this study is to investigate the expression of brachyury expression in a series of chordomas in conjunction to clinicopathological characteristics and to review the relevant literature providing all the details needed in the immunohistochemical study of brachyury. MATERIALS AND METHODS: This is a retrospective study of 62 chordomas, diagnosed over a 22-year period. No dedifferentiated or poorly differentiated cases were included. A monoclonal primary antibody (clone A-4) was used and brachyury expression was evaluated by the H-score. Clinicopathological parameters studied were age, sex, tumor localization, decalcification status and tissue age. Fetal notochords were used for comparison. RESULTS: Mean H-score of nuclear brachyury expression was 129.8. The tissue age significantly influenced brachyury expression, the older samples expressing less brachyury. Decalcification demonstrated a trend to weaken brachyury expression. Clinical characteristics were not correlated with the patterns of brachyury expression. Notochords were negative. Literature review reveals several polyclonal antibodies used and a positivity of 75%-100% in chordomas with even more variable results in notochords. CONCLUSION: In chordomas, as in other tumor types, an uniformization of studies about brachyury expression is needed, by considering the clone used, and the decalcification and the age of the sample, given the growing importance of brachyury in diagnosis and therapeutic steps.

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.

High endothelial venules are present in pharyngeal and laryngeal carcinomas and they are associated with better prognosis.

BACKGROUND: Tumors lymphocytic infiltration has prognostic and predictive value. However, the mechanisms involved in lymphocyte recruitment remain poorly characterized. High endothelial venules (HEV) are blood vessels specialized in lymphocyte recruitment, recently showing prognostic significance in some types of cancer. Their implications in laryngeal or pharyngeal cancer is largely unknown. AIM OF THE STUDY: To investigate the possible presence of HEVs in head and neck cancer. MATERIAL AND METHODS: Oropharyngeal (n = 61), hypopharyngeal (n = 53) and laryngeal (n = 21) squamous cell carcinomas were immunohistochemically studied with the MECA-79 antibody, which specifically recognizes HEVs. Histological and clinical factors were correlated with HEVs' presence. RESULTS: HEVs were present in 34% of tumors, showing significant correlations with oropharyngeal localization, higher lymphocytic response, lower tumor budding, lower T status, absence of distant metastases and better overall and progression-free survival. CONCLUSION: HEVs represent an important prognostic factor in head and neck cancer.

INSM1 Expression in Chordomas.

OBJECTIVES: Chordomas are rare malignant tumors with a broad differential diagnosis, including chondrosarcomas and metastatic carcinomas. Recently, insulinoma-associated protein 1 (INSM1) has gained great interest regarding the diagnosis of neuroendocrine tumors but also extraskeletal myxoid chondrosarcomas. However, its expression in chordomas remains largely unknown. METHODS: We retrospectively examined 57 chordomas for INSM1 expression. RESULTS: INSM1 expression was found in only 5% of tumors. CONCLUSIONS: This marker is rarely expressed in this type of tumor, raising questions about neuroendocrine differentiation.

Meningioma sampling: how much is enough for the accurate grading of atypical meningiomas?

Meningioma grading relies on several pathological criteria (brain invasion, mitotic count, sheeting, small cell foci, necrosis, macronucleoli and hypercellularity) and histopathological subtypes. Regardless of histopathological subtype, the presence of these pathological parameters can be focally present and not present on each slide of a meningioma. We performed (1) a retrospective work comparing the frequency of parameters used for meningioma grading between two periods with different sampling techniques, and (2) we calculated the probability of presence of each criterion on resected meningiomas entirely processed included and examined. First, we compared two time periods: between 2002-2008 where meningiomas were not all entirely sampled, and between 2012-2018 where all meningiomas were entirely sampled. The frequency of tumour grades was not significantly different between the two periods (p=0.17). Mitosis ≥4/1.6mm2, small cell foci, macronucleoli and hypercellularity were more frequently found when meningiomas were entirely sampled (p<0.05). Second, we focused on 59 grade 2 meningiomas entirely sampled to highlight the distribution of histopathological parameters used for meningioma grading. We have shown that a correct grading of more than 95% of meningiomas can be achieved when at least six slides are examined. Our work suggests that meningioma sampling might be an issue and the sampling system must be specified in research works on grading.