CytoLyt fixation impedes insulinoma-associated protein 1 (INSM1) immunoreactivity compared to formalin fixation.

INTRODUCTION: Insulinoma-associated protein 1 (INSM1) is an immunohistochemical marker commonly used to confirm cytomorphological concordant neuroendocrine tumors/carcinomas (NETs/NECs), demonstrating high utility in small samples. Previous reports have suggested comparable INSM1 staining in CytoLyt-fixed cell blocks and formalin-fixed surgical pathology specimens. This study aimed to assess INSM1 immunoreactivity using both fixation methods and investigate potential factors contributing to its variable expression. MATERIALS AND METHODS: A retrospective query was performed (03/31/21-05/31/22) for NET/NEC cases that had both formalin- and CytoLyt-fixed cell blocks. We collected clinical data and reporting of immunostains for each case. INSM1 staining was evaluated in both fixation methods, and reported as positive, negative, or equivocal. Equivocal INSM1 staining was further scored as a percentage of 1%-100% and intensity of weak (faint staining), moderate (darker staining), and strong (dense staining). RESULTS: Our search identified 20 cases from diverse body sites, including mediastinal lymph nodes (40%), pancreas (35%), lung (20%), and porta hepatis lymph nodes (5%). All cases exhibited a widespread positivity (over 90%) in formalin-fixed cell blocks. In contrast, CytoLyt fixed cells showed a negative stain in 65% of cases and 30% exhibited an equivocal positivity. CONCLUSIONS: While INSM1 is previously reported as a sensitive (75%-100%) and specific (82.7%-100%) marker for NET/NECs, our study found a reduced immunohistochemical staining in CytoLyt-fixed cell blocks. Consequently, false negative INSM1 immunohistochemical results in CytoLyt-fixed cell block material may pose a pitfall in the diagnosis of NET/NEC.

Clonal Origin and Lineage Ambiguity in Mixed Neuroendocrine Carcinoma of the Uterine Cervix.

Small-cell neuroendocrine carcinoma (SCNEC) of the cervix is a rare disease characterized by a high incidence of mixed tumors with other types of cancer. The mechanism underlying this mixed phenotype is not well understood. This study established a panel of organoid lines from patients with SCNEC of the cervix and ultimately focused on one line, which retained a mixed tumor phenotype, both in vitro and in vivo. Histologically, both organoids and xenograft tumors showed distinct differentiation into either SCNEC or adenocarcinoma in some regions and ambiguous differentiation in others. Tracking single cells indicated the existence of cells with bipotential differentiation toward SCNEC and adenocarcinomas. Single-cell transcriptional analysis identified three distinct clusters: SCNEC-like, adenocarcinoma-like, and a cluster lacking specific differentiation markers. The expression of neuroendocrine markers was enriched in the SCNEC-like cluster but not exclusively. Human papillomavirus 18 E6 was enriched in the SCNEC-like cluster, which showed higher proliferation and lower levels of the p53 pathway. After treatment with anticancer drugs, the expression of adenocarcinoma markers increased, whereas that of SCNEC decreased. Using a reporter system for keratin 19 expression, changes in the differentiation of each cell were shown to be associated with the shift in differentiation induced by drug treatment. These data suggest that mixed SCNEC/cervical tumors have a clonal origin and are characterized by an ambiguous and flexible differentiation state.

Druggable growth dependencies and tumor evolution analysis in patient-derived organoids of neuroendocrine neoplasms from multiple body sites.

Neuroendocrine neoplasms (NENs) comprise well-differentiated neuroendocrine tumors (NETs) and poorly differentiated neuroendocrine carcinomas (NECs). Treatment options for patients with NENs are limited, in part due to lack of accurate models. We establish patient-derived tumor organoids (PDTOs) from pulmonary NETs and derive PDTOs from an understudied subtype of NEC, large cell neuroendocrine carcinoma (LCNEC), arising from multiple body sites. PDTOs maintain the gene expression patterns, intra-tumoral heterogeneity, and evolutionary processes of parental tumors. Through hypothesis-driven drug sensitivity analyses, we identify ASCL1 as a potential biomarker for response of LCNEC to treatment with BCL-2 inhibitors. Additionally, we discover a dependency on EGF in pulmonary NET PDTOs. Consistent with these findings, we find that, in an independent cohort, approximately 50% of pulmonary NETs express EGFR. This study identifies an actionable vulnerability for a subset of pulmonary NETs, emphasizing the utility of these PDTO models.

Small Cell Neuroendocrine Carcinoma of the Parotid Gland: An Uncommon Example of Incompletely Encapsulated Tumor Including S100 Protein-Positive Clear Cells.

Small cell carcinoma is rare in salivary glands and has recently been termed small cell neuroendocrine carcinoma. We herein describe an uncommon example arising in the parotid gland. The patient was a 75 yearold Japanese male who had swelling in the right parotid area. He underwent a superficial lobectomy and, after a histological diagnosis was made, a total parotidectomy. Histologically, the tumor had a thick hyalinized capsule that was incomplete, beyond which the tumor invaded into the surrounding parotid parenchyma. The tumor consisted of typical small basophilic cells intermingled with bland clear cells, between which a gradual transition was observed both inside and outside the capsule. Small basophilic cells were immunoreactive for chromograninA as well as synaptophysin, while clear cells were positive for S100 protein. The Ki-67 labeling rate reached 30-40% at the high points of small basophilic cells, but clear cells were minimally labelled. The present case was considered a dedifferentiated carcinoma of the parotid gland, possibly with acinic cell carcinoma as a precursor. This tumor could also be considered a "mixed exocrine-endocrine carcinoma," which may explain the histogenesis of neuroendocrine carcinomas in non-endocrine organs that are not included in the diffuse (dispersed) neuroendocrine system, such as the parotid gland.

PAX1 expression in thymic epithelial neoplasms and morphologic mimics.

Thymic epithelial neoplasms are morphologically diverse and can pose a diagnostic challenge that is complicated by a lack of immunohistochemistry (IHC) markers that are entirely sensitive and specific for thymic epithelium. Polyclonal PAX8 is often used in this context, but it is not a specific marker. The PAX1 transcription factor shares significant homology with PAX8 and plays an integral role in thymic development in humans and murine models. This study evaluated the role of PAX1 IHC in differentiating thymic epithelial neoplasms from morphologic mimics on whole slide tissue sections. The PAX1 antibody stained all 74 thymoma cases; however, there was wide variability in staining intensity within each subtype. The antibody was less sensitive in thymic carcinomas and thymic neuroendocrine tumors compared to thymomas and demonstrated weak staining in a subset of morphologic mimics (21 squamous cell carcinomas, 6 pulmonary neuroendocrine tumors, 1 mesothelioma, 1 lymphoblastic lymphoma, and 1 granulosa cell tumor). With a H-score positive threshold of 75, the antibody had 100% specificity, and sensitivities of 92%, 56%, and 47% in thymomas, thymic neuroendocrine tumors, and thymic carcinomas respectively. The PAX1 antibody showed frequent geographic reduction in staining consistent with compromised antigenicity from variable formalin fixation. PAX1 IHC has a moderate-to-high sensitivity for thymic epithelial neoplasms; however, the wide staining variability and fixation effects may lead to difficulty with consistent interpretation. This marker is unlikely to supplant the role of PAX8 in diagnostic practice, but it may be a useful addition to immunohistochemistry panels when evaluating for thymic primary tumors.

[Small cell neuroendocrine carcinoma of the endometrium]. / Melkokletochnaya neiroendokrinnaya kartsinoma endometriya.

The article describes a rare case of small cell neuroendocrine carcinoma of the endometrium in a 67-year-old woman. According to the literature, only about 90 such observations have been described worldwide. Histological examination revealed three necessary features: the small-cell nature of the tumor, the presence of epithelial and neuroendocrine markers. An IHC study revealed a positive expression of Syn, Chrom A, CD56, CK AE1/AE3 markers; the proliferative activity index was 70%.

[Clinicopathological Features of Primary Small Cell Neuroendocrine Carcinoma of the Bladder].

Objective To investigate the clinicopathological features,immunohistochemical features,diagnosis,and relationship with sporadic prostate cancer in primary small cell neuroendocrine carcinoma of the bladder. Methods We retrospectively analyzed the clinical characteristics of 12 patients with primary small cell neuroendocrine carcinoma of the bladder diagnosed at Beijing Chao-Yang Hospital affiliated to Capital Medical University from January 2013 to September 2022.The histological features of primary small cell neuroendocrine carcinoma of the bladder were re-evaluated by two pathologists according to the 2022 revision of the World Health Organization Classification of Tumors of the Urinary System and Male Genital Organs.Electronic medical records were retrieved,and telephone follow-up was conducted from the time of histopathological diagnosis to the death or the end of the last follow-up until January 31,2023. Results The 12 patients include 7 patients in pT3 stage and 1 patient in pT4 stage.Eight patients were complicated with other types of tumors,such as high-grade urothelial carcinoma of the bladder and squamous cell carcinoma.Five patients had sporadic prostate cancer.Immunohistochemical staining showed that 12 (100.0%),10 (83.3%),and 8 (66.7%) patients were tested positive for CD56,Syn,and CgA,respectively.The Ki67 proliferation index ranged from 80% to 90%.Five patients with urothelial carcinoma were tested positive for CK20,GATA3,and CK7.P504S was positive in all the 5 patients with prostate cancer,while P63 and 34ßE12 were negative.The follow-up of the 12 patients lasted for 3-60 months.Eight of these patients died during follow-up,with the median survival of 15.5 months.Four patients survived. Conclusions Primary small cell neuroendocrine carcinoma of the bladder is a rare urological tumor with high aggressiveness and poor prognosis.In male patients with bladder prostatectomy,all prostate tissue should be sampled.If prostate cancer is detected,the prostate-specific antigen level should be monitored.

An integrated cellular and molecular model of gastric neuroendocrine cancer evolution highlights therapeutic targets.

Gastric neuroendocrine carcinomas (G-NEC) are aggressive malignancies with poorly understood biology and a lack of disease models. Here, we use genome sequencing to characterize the genomic landscapes of human G-NEC and its histologic variants. We identify global and subtype-specific alterations and expose hitherto unappreciated gains of MYC family members in a large part of cases. Genetic engineering and lineage tracing in mice delineate a model of G-NEC evolution, which defines MYC as a critical driver and positions the cancer cell of origin to the neuroendocrine compartment. MYC-driven tumors have pronounced metastatic competence and display defined signaling addictions, as revealed by large-scale genetic and pharmacologic screening of cell lines and organoid resources. We create global maps of G-NEC dependencies, highlight critical vulnerabilities, and validate therapeutic targets, including candidates for clinical drug repurposing. Our study gives comprehensive insights into G-NEC biology.

Intratumoral dendritic cells and T cells predict survival in gastroenteropancreatic neuroendocrine neoplasms.

Clinical management of gastroenteropancreatic neuroendocrine neoplasms remains challenging. We recently introduced the FMS-like tyrosine kinase 3 ligand (FLT3LG) as a possible biomarker for a proinflammatory tumor microenvironment. Here, we put a spotlight on the quantitative assessment of classical dendritic cells (cDC) and T cells in the context of FLT3LG mRNA levels in a retrospective study on neuroendocrine tumor (NET) G2/G3 and neuroendocrine carcinoma (NEC) of pancreatic and gastric origin. The abundance of cDC and T cells and their relevant subpopulations were determined by immunofluorescent staining and correlated with FLT3LG mRNA levels as well as clinical outcomes. Immune cell counts attested to highly variable infiltration densities. Samples with the presence of cDC or high numbers of T cells exhibited increased FLT3LG expression. Abundance of cDC, defined as HLA-DR+CD11c+ cells with CLEC9a (cDC1) or CD1c (cDC2), as well as T cells correlated with FLT3LG mRNA levels and predicted disease-specific survival. Combining FLT3LG and T cell counts further improved this prediction. Therefore, tumor-infiltrating cDC and T cells are prognostic markers in NET G2/G3 or NEC and FLT3LG mRNA may serve as a simple-to-use biomarker for a quantitative estimate of their abundance, mandating prospective evaluation in the context of immune-targeted therapies.

The Approach of Artificial Intelligence in Neuroendocrine Carcinomas of the Breast: A Next Step towards Precision Pathology?-A Case Report and Review of the Literature.

Primary neuroendocrine tumors (NETs) of the breast are considered a rare and undervalued subtype of breast carcinoma that occur mainly in postmenopausal women and are graded as G1 or G2 NETs or an invasive neuroendocrine carcinoma (NEC) (small cell or large cell). To establish a final diagnosis of breast carcinoma with neuroendocrine differentiation, it is essential to perform an immunohistochemical profile of the tumor, using antibodies against synaptophysin or chromogranin, as well as the MIB-1 proliferation index, one of the most controversial markers in breast pathology regarding its methodology in current clinical practice. A standardization error between institutions and pathologists regarding the evaluation of the MIB-1 proliferation index is present. Another challenge refers to the counting process of MIB-1's expressiveness, which is known as a time-consuming process. The involvement of AI (artificial intelligence) automated systems could be a solution for diagnosing early stages, as well. We present the case of a post-menopausal 79-year-old woman diagnosed with primary neuroendocrine carcinoma of the breast (NECB). The purpose of this paper is to expose the interpretation of MIB-1 expression in our patient' s case of breast neuroendocrine carcinoma, assisted by artificial intelligence (AI) software (HALO-IndicaLabs), and to analyze the associations between MIB-1 and common histopathological parameters.

Advances in the Treatment of Gastroenteropancreatic Neuroendocrine Carcinomas: Are we Moving Forward?

Poorly differentiated gastroenteropancreatic neuroendocrine carcinomas are aggressive neoplasms of challenging clinical management. A small proportion of patients with early-stage disease may achieve long-term survival, but the majority of patients present with rapidly lethal metastatic disease. Current standard of care still follows the treatment paradigm of small cell lung cancer, a far more common G3 neuroendocrine neoplasm, although emerging molecular and clinical data increasingly question this approach. In this article, we will briefly summarize epidemiology and prognosis of gastroenteropancreatic neuroendocrine carcinomas to emphasize the very low incidence, aggressive nature, and orphan status of this tumor entity. We will also discuss the current pathological classification and its limitations, as well as recent data on their differential biological background compared with small cell lung cancer, and its potential implications for patients care. Then, we will review the standard of care of systemic therapy, basically focused on platinum-based cytotoxic chemotherapy, including some recent randomized trials providing evidence regarding efficacy of irinotecan vs etoposide platinum doublets. Finally, we will present a comprehensive overview of novel therapeutic strategies in current clinical development, including recently reported data on immunotherapy, tumor-agnostic therapies (microsatellite instability, high tumor mutational burden, NTRK and RET gene fusions, BRAF or KRAS inhibitors), and additional treatment strategies targeting other tumor vulnerabilities (ie, Notch pathway, novel targets for radioligand therapy), and provide some insights regarding unmet needs and future perspectives to improve patient's care and prognosis.

[Clinical analysis of 11 patients with neuroendocrine carcinoma in maxillofacial region].

Objective: To investigate the clinicopathological features, treatment and prognosis of maxillofacial neuroendocrine carcinoma. Methods: A total of 11 patients with maxillofacial neuroendocrine carcinoma diagnosed in the Department of Pathology of The First Affiliated Hospital of Zhengzhou University from December 2010 to July 2022 were retrospectively enrolled, including 8 males and 3 females, aged (65.2±9.5) years (ranged from 49 to 87 years), with a disease course of 0.5 to 6.0 months. The clinicopathological data including head and neck CT, MRI and treatment methods were analyzed. Results: Submandibular gland and maxilla were involved in 3 cases, parapharynx in 2 cases, and face, tongue root and soft palate in 1 case respectively. Clinically, the initial symptom is a rapidly growing painless or tender mass, which may be accompanied by restricted mouth opening, dysphagia, and local numbness after invasion of masticatory muscles and nerves. The tumors were all invasive and low-density, with unclear boundaries from the surrounding tissues. Among the patients, 9 received surgical treatment, and 5 received adjuvant treatment after surgery (2 received chemotherapy, 3 received radiotherapy+chemotherapy). According to the 5th edition of the World Health Organization classification of head and neck tumors in 2022, there were 1 case (1/11) with poorly differentiated large cells and 10 cases (10/11) with poorly differentiated small cells. Histologically, the macrocell type is composed of large cells with rough chromatin, obvious vacuolar nucleolus, protruding nucleolus, and necrosis. The small cell type is dominated by small blue round cells with neuroendocrine characteristics, with active growth and multifocal necrosis. Immunohistochemical staining showed that cytokeratin (CK), epithelial membrane antigen (EMA) and synaptophysin (Syn) were diffusively expressed, 10 cases expressed CD56, 8 cases expressed p63, 6 cases expressed weakly punctated chromograin-A (CgA), and S-100 was not expressed. The Ki-67 index ranges from 20 to 90 percent. By the end of follow-up (0.5 to 127.0 months), 3 patients were alive, and the mean progression-free survival (21.0 months) of postoperative chemoradiotherapy patients was significantly longer than that of surgery and/or chemotherapy alone (3.3 months). Conclusions: Maxillofacial neuroendocrine carcinoma is characterized by low differentiation of small cells, high degree of malignancy and poor prognosis. Radical surgery combined with chemoradiotherapy has better local control effect.

Dependence on MUC1-C in Progression of Neuroendocrine Prostate Cancer.

Castration resistant prostate cancer (CRPC) is responsive to androgen receptor (AR) axis targeted agents; however, patients invariably relapse with resistant disease that often progresses to neuroendocrine prostate cancer (NEPC). Treatment-related NEPC (t-NEPC) is highly aggressive with limited therapeutic options and poor survival outcomes. The molecular basis for NEPC progression remains incompletely understood. The MUC1 gene evolved in mammals to protect barrier tissues from loss of homeostasis. MUC1 encodes the transmembrane MUC1-C subunit, which is activated by inflammation and contributes to wound repair. However, chronic activation of MUC1-C contributes to lineage plasticity and carcinogenesis. Studies in human NEPC cell models have demonstrated that MUC1-C suppresses the AR axis and induces the Yamanaka OSKM pluripotency factors. MUC1-C interacts directly with MYC and activates the expression of the BRN2 neural transcription factor (TF) and other effectors, such as ASCL1, of the NE phenotype. MUC1-C also induces the NOTCH1 stemness TF in promoting the NEPC cancer stem cell (CSC) state. These MUC1-C-driven pathways are coupled with activation of the SWI/SNF embryonic stem BAF (esBAF) and polybromo-BAF (PBAF) chromatin remodeling complexes and global changes in chromatin architecture. The effects of MUC1-C on chromatin accessibility integrate the CSC state with the control of redox balance and induction of self-renewal capacity. Importantly, targeting MUC1-C inhibits NEPC self-renewal, tumorigenicity and therapeutic resistance. This dependence on MUC1-C extends to other NE carcinomas, such as SCLC and MCC, and identify MUC1-C as a target for the treatment of these aggressive malignancies with the anti-MUC1 agents now under clinical and preclinical development.

Matrix Metalloproteinases 2 and 9 and Their Tissue Inhibitors in the Diagnostics of Medullary Thyroid Carcinoma.

Medullary Thyroid Carcinoma (MTC) is a tumor of the neuroendocrine system. In recent years, the need to assess the MTC diagnostic-related parameters has emerged with the aim to elucidate the mechanisms involved in this pathology. The objective of this study was to evaluate the role of Matrix Metalloproteinases (MMPs) 2 and 9, their tissue inhibitors of matrix metalloproteinases (TIMPs), S100 protein, and amyloid in the diagnostic of MTC. Thirty-two samples with MTC (72% women) were included in this cross-sectional study and divided by groups: T category 1 (T1)≤20 mm and T category 2 (T2) 20 to 40 mm of tumor size. MMPs 2 and 9, TIMPs 2 and 1, S100 protein, and calcitonin in tissues were obtained by immunohistochemical techniques. The presence of amyloid in tissue sections was detected on Thioflavin T-stained slides under fluorescent microscope. Percentage of positive cells (P) observed for MMP-2 was higher in those samples presenting T2 MTC with respect to those with T1 MTC ( P <0.05). Moreover, P-MMP-2 showed a direct correlation with higher T category of MTC (Rho=0.439, P < 0.001), whereas P-MPP-9 was directly correlated with S100 protein and the intensity of calcitonin in tissues (Rho=0.419, P =0.017; Rho=0.422, P =0.016, respectively. Therefore, MMPs were directly correlated with some traditional biomarkers of MTC. In this regard, P-MMP-2 was more expressed in type 2 MTC. Combining the analysis of traditional and other useful biomarkers of MTC as MMPs 2 and 9 could be a useful strategy in the diagnostic of MTC.

Pulmonary cancers across different histotypes share hybrid tuft cell/ionocyte-like molecular features and potentially druggable vulnerabilities.

Tuft cells are chemosensory epithelial cells in the respiratory tract and several other organs. Recent studies revealed tuft cell-like gene expression signatures in some pulmonary adenocarcinomas, squamous cell carcinomas (SQCC), small cell carcinomas (SCLC), and large cell neuroendocrine carcinomas (LCNEC). Identification of their similarities could inform shared druggable vulnerabilities. Clinicopathological features of tuft cell-like (tcl) subsets in various lung cancer histotypes were studied in two independent tumor cohorts using immunohistochemistry (n = 674 and 70). Findings were confirmed, and additional characteristics were explored using public datasets (RNA seq and immunohistochemical data) (n = 555). Drug susceptibilities of tuft cell-like SCLC cell lines were also investigated. By immunohistochemistry, 10-20% of SCLC and LCNEC, and approximately 2% of SQCC expressed POU2F3, the master regulator of tuft cells. These tuft cell-like tumors exhibited "lineage ambiguity" as they co-expressed NCAM1, a marker for neuroendocrine differentiation, and KRT5, a marker for squamous differentiation. In addition, tuft cell-like tumors co-expressed BCL2 and KIT, and tuft cell-like SCLC and LCNEC, but not SQCC, also highly expressed MYC. Data from public datasets confirmed these features and revealed that tuft cell-like SCLC and LCNEC co-clustered on hierarchical clustering. Furthermore, only tuft cell-like subsets among pulmonary cancers significantly expressed FOXI1, the master regulator of ionocytes, suggesting their bidirectional but immature differentiation status. Clinically, tuft cell-like SCLC and LCNEC had a similar prognosis. Experimentally, tuft cell-like SCLC cell lines were susceptible to PARP and BCL2 co-inhibition, indicating synergistic effects. Taken together, pulmonary tuft cell-like cancers maintain histotype-related clinicopathologic characteristics despite overlapping unique molecular features. From a therapeutic perspective, identification of tuft cell-like LCNECs might be crucial given their close kinship with tuft cell-like SCLC.

FOXA2 drives lineage plasticity and KIT pathway activation in neuroendocrine prostate cancer.

Prostate cancer adeno-to-neuroendocrine lineage transition has emerged as a mechanism of targeted therapeutic resistance. Identifying the direct molecular drivers and developing pharmacological strategies using clinical-grade inhibitors to overcome lineage transition-induced therapeutic resistance are imperative. Here, using single-cell multiomics analyses, we investigate the dynamics of cellular heterogeneity, transcriptome regulation, and microenvironmental factors in 107,201 cells from genetically engineered mouse prostate cancer samples with complete time series of tumor evolution seen in patients. We identify that FOXA2 orchestrates prostate cancer adeno-to-neuroendocrine lineage transition and that Foxa2 expression is significantly induced by androgen deprivation. Moreover, Foxa2 knockdown induces the reversal of adeno-to-neuroendocrine transition. The KIT pathway is directly regulated by FOXA2 and specifically activated in neuroendocrine prostate cancer (NEPC). Pharmacologic inhibition of KIT pathway significantly suppresses mouse and human NEPC tumor growth. These findings reveal that FOXA2 drives adeno-to-neuroendocrine lineage plasticity in prostate cancer and provides a potential pharmacological strategy for castration-resistant NEPC.

Detection of HPV RNA in Extracellular Vesicles from Neuroendocrine Cervical Cancer Cells.

Background: Neuroendocrine carcinoma of the cervix (NECC) is an aggressive and rare type of cervical cancer. The five-year overall survival is low at 30% and there is no standardized therapy based on controlled trials for this type of tumour. Most are locally advanced or metastasized at the time of the diagnosis. Extracellular vesicles (EVs) could be a carrier of viral DNA/RNA, given their vital role in cellular communication. The content of EV derived from NECC cells has not been investigated due to the lack of cell line, and it is not known whether they contain human papillomaviruses (HPV) DNA/RNA or not. Methods: The presence of viral E7 DNA/RNA in EVs purified from a culture of a recently established NECC cell line, GUMC-395, was evaluated by using droplet digital polymerase chain reaction (ddPCR). These EVs were characterized using nanoparticle tracking analysis (NTA) for size distribution, transmission electron microscopy (TEM) for morphology, Western blot for CD63, and bioanalyser for RNA quantity and quality. Results: HPV16 viral-RNA, but not DNA, was detected in EVs from GUMC-395 using ddPCR. NTA identified EVs with a mean diameter of 105.0 nm, TEM confirmed normal morphological shape and size, and Western blot analysis confirmed the presence of EV-associated proteins CD63. The EVs were found to be enriched with small RNAs using a bioanalyser. Conclusions: HPV16 RNA is found in EVs from a neuroendocrine cervical cancer and could be involved in the pathogenesis of the disease and used as a diagnostic biomarker.

Comprehensive analysis of clinicopathologic features and p53 mutation in neuroendocrine neoplasms of the breast: experience from a large academic center.

PURPOSE: The recent WHO classification of breast cancer (2019) categorizes breast carcinoma with neuroendocrine (NE) differentiation into three morphologically distinct subtypes: well-differentiated neuroendocrine tumor (NET), poorly differentiated neuroendocrine carcinoma (NEC), and invasive breast carcinoma, no special type with neuroendocrine differentiation (IBC-NST-NE). Data regarding the prognostic significance of neuroendocrine differentiation are conflicting and an association, if any, between p53 mutation and neuroendocrine differentiation is largely unknown. METHODS: We examined p53 expression and other clinicopathologic characteristics in three types of invasive breast carcinoma with NE differentiation in a cohort of sixty-three patients, including 45 IBC-NST with NE differentiation, 10 NETs, and 8 NECs. RESULTS: No significant difference of clinicopathologic feature was observed between IBC-NST with NE differentiation and NET, but NECs showed significantly lower expressions of hormone receptors, more mutated p53, and higher frequency of distant metastases than IBC-NST with NE differentiation and NETs. CONCLUSION: NECs of the breast are genetically and clinically different from IBC-NST-NEs and NETs of the breast.

Integrated pathological analysis to develop a Gal-9 based immune survival stratification to predict the outcome of lung large cell neuroendocrine carcinoma and its usefulness in immunotherapy.

This study aimed to integrate the cell spatial organization to develop a Gal-9-based immune survival stratification in the lung large cell neuroendocrine carcinoma (LCNEC) and investigate its potentials to immunotherapy. The expression of Gal-9 and other twelve immune markers were evaluated in 122 cases of surgical LCNEC samples from our center using immunohistochemistry. The Gal-9-based immune survival stratification risk score was constructed and its predictive performance was evaluated. Then, we thoroughly explored the effects of Gal-9 and immune risk score on LCNEC immune pathways, immune micro-environment and immunotherapy sensitivity in different cohort and platform, and made a validation in pathology images using Histology-based Digital-Staining (HDS). In 122 LCNEC samples, 43 cases were positive Gal-9 expression on tumor cells (Gal-9 TC). Increased Gal-9 TC predicted worse overall survival. Gal-9's interaction with other immune markers added to the immune suppression and immune tolerance in LCNEC. Immune protein marker-based risk score consisting of Gal-9, CD3, CD4, PD-L1, and PD-1 was developed and validated to robustly discriminate survival high-risk or low-risk in LCNEC patients. The high-risk group characterized by immune-desert tumor had less various T cells. The low-risk group featuring immune-inflamed tumor was more likely to respond to anti-PD1 immunotherapy. HDS in 122 LCNEC samples' 108,369 cells validated that the high-risk group had more tumor cells, less stromal cells, less lymphocytes, higher tumor cell nucleic solidity and lower stromal cells nucleic solidity. An integrated pathological analysis confirms the Gal-9 based immune survival stratification is distinctively related to micro-environment status involved in immune suppression and immune tolerance and could act as a combinatorial biomarker to predict the outcome of LCNEC. These findings may help effectively stratify LCNEC patients sensitive to immunotherapy.

Genetics of neuroendocrine prostate cancer: recent progress in genetic understanding is translating into therapeutic opportunities.

PURPOSE OF REVIEW: Neuroendocrine prostate cancer (NEPC) is a rare histologic subtype of prostate cancer with extremely aggressive clinical behaviour and very limited data regarding treatment options. This review is intended to relay new research advances in the understanding of the genetic and epigenetic aberrations underlying NEPC development and to review new targeted therapeutic options developed based on NEPC genetics. RECENT FINDINGS: Multiple genomic alterations and epigenetic regulators have been identified in NEPC development. Among these are amplifications of oncogenic transcriptional factors, changes in expression of cell surface markers and epigenetic alterations. This in turn has facilitated a number of new targeted therapies for NEPC that act via different mechanisms including catalytic inhibitors, immune-modulators and epigenetic modifiers. These targeted therapies are now being studied in different phases of clinical trials with some preliminary results showing efficacy. SUMMARY: NEPC is a highly aggressive malignancy with currently lack of effective treatments. Considerable challenges still remains to improve clinical outcomes in NEPC; however, ongoing trials exploiting novel genetic and epigenetic alterations hold promise for patients suffering from this aggressive disease.