Sequential Colocalization of ERa, PR, and AR Hormone Receptors Using Confocal Microscopy Enables New Insights into Normal Breast and Prostate Tissue and Cancers

Multiplex immunohistochemistry (mIHC) use markers staining different cell populations applying widefield optical microscopy. Resolution is low not resolving subcellular co-localization. We sought to colocalize markers at subcellular level with antibodies validated for clinical diagnosis, including the single secondary antibody (combination of anti-rabbit/mouse-antibodies) used for diagnostic IHC with any primary antibody, and confocal microscopy. We explore colocalization in the nucleus (ColNu) of nuclear hormone receptors (ERa, PR, and AR) along with the baseline marker p63 in paired samples of breast and prostate tissues. We established ColNu mIHCF as a reliable technique easily implemented in a hospital setting. In ERa+ breast cancer, we identified different colocalization patterns (nuclear or cytoplasmatic) with PR and AR on the luminal epithelium. A triple-negative breast-cancer case expressed membrane-only ERa. A PR-only case was double positive PR/p63. In normal prostate, we identified an ERa+/p63+/AR-negative distinct population. All prostate cancer cases characteristically expressed ERa on the apical membrane of the AR+ epithelium. We confirmed this using ERa IHC and needle-core biopsies. ColNu mIHCF is feasible and already revealed a new marker for prostate cancer and identified sub-patterns in breast cancer. It could be useful for pathology as well as for functional studies in normal prostate and breast tissues

Metastases of renal cell carcinoma to the thyroid gland with synchronous benign and malignant follicular cell-derived neoplasms

Clear cell renal cell carcinoma (CCRCC) is the most common origin for metastasis in the thyroid. A 51-year-old woman was referred to our hospital for a subcarinal lesion. Ten years before, the patient had undergone a nephrectomy for CCRCC. Whole-body fluorodeoxyglucose positron emission tomography revealed elevated values in the thyroid gland, while the mediastinum was normal. An endoscopic ultrasonography-guided fine-needle aspiration biopsy of the mediastinal mass was consistent with CCRCC, and this was confirmed after resection. The thyroidectomy specimen also revealed lymphocytic thyroiditis, nodular hyperplasia, one follicular adenoma, two papillary microcarcinomas, and six foci of metastatic CCRCC involving both thyroid lobes. Curiously two of the six metastatic foci were located inside two adenomatoid nodules (tumor-in-tumor). The metastatic cells were positive for cytokeratins, CD10, epidermal growth factor receptor, and vascular endothelial growth factor receptor 2. No BRAF gene mutations were found in any of the primary and metastatic lesions. The patient was treated with sunitinib and finally died due to CCRCC distant metastases 6 years after the thyroidectomy. In CCRCC patients, a particularly prolonged survival rate may be achieved with the appropriate therapy, in contrast to the ominous prognosis typically found in patients with thyroid metastases from other origins

Thyroid Pathology Findings in Cowden Syndrome: A Clue for the Diagnosis of the PTEN Hamartoma Tumor Syndrome

OBJECTIVES: PTEN hamartoma tumor syndrome (PHTS) is a hereditary disorder caused by germline inactivating mutations of the PTEN gene. PHTS includes Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome. We describe how the peculiar pathologic and immunohistochemical thyroid features lead pathologists to suggest PHTS. METHODS: A 28-year-old white Spanish woman had a multinodular goiter. Total thyroidectomy was performed after fine-needle aspiration biopsy. Microscopic, immunohistochemical, and molecular analyses of the thyroid lesions were realized. RESULTS: The thyroid was multinodular, showing one papillary microcarcinoma, five follicular adenomas, three adenolipomas, 46 tiny adenomatous nodules (microadenomas), scattered foci of adipose tissue, and lymphocytic thyroiditis. Tumors were positive for thyroglobulin, thyroperoxidase, pendrin, cyclin D1, and p27 but negative for calcitonin and PTEN. A germline heterozygous deletion of one adenine at nucleotide 827 in exon 8 of the PTEN gene was confirmed. No BRAF, NRAS, or KRAS somatic mutations were detected in the papillary microcarcinoma, follicular adenoma, adenolipomas, or microadenomas. Negativity for PTEN was also found in the colonic tubulovillous adenoma and the storiform collagenoma. CONCLUSIONS: Pathologists play a crucial role in recognizing pathologic thyroid findings associated with PHTS for selecting patients for genetic testing

Chromosomal, epigenetic and microRNA-mediated inactivation of LRP1B, a modulator of the extracellular environment of thyroid cancer cells

The low-density lipoprotein receptor-related protein (LRP1B), encoding an endocytic LDL-family receptor, is among the 10 most significantly deleted genes across 3312 human cancer specimens. However, currently the apparently crucial role of this lipoprotein receptor in carcinogenesis is not clear. Here we show that LRP1B inactivation (by chromosomal, epigenetic and microRNA (miR)-mediated mechanisms) results in changes to the tumor environment that confer cancer cells an increased growth and invasive capacity. LRP1B displays frequent DNA copy number loss and CpG island methylation, resulting in mRNA underexpression. By using CpG island reporters methylated in vitro, we found that DNA methylation disrupts a functional binding site for the histone-acetyltransferase p300 located at intron 1. We identified and validated an miR targeting LRP1B (miR-548a-5p), which is overexpressed in cancer cell lines as a result of 8q22 DNA gains. Restoration of LRP1B impaired in vitro and in vivo tumor growth, inhibited cell invasion and led to a reduction of matrix metalloproteinase 2 in the extracellular medium. We emphasized the role of an endocytic receptor acting as a tumor suppressor by modulating the extracellular environment composition in a way that constrains the invasive behavior of the cancer cells

A Novel Nanoproteomic Approach for the Identification of Molecular Targets Associated with Thyroid Tumors

A thyroid nodule is the most common presentation of thyroid cancer; thus, it is extremely important to differentiate benign from malignant nodules. Within malignant lesions, classification of a thyroid tumor is the primary step in the assessment of the prognosis and selection of treatment. Currently, fine-needle aspiration biopsy (FNAB) is the preoperative test most commonly used for the initial thyroid nodule diagnosis. However, due to some limitations of FNAB, different high-throughput "omics" approaches have emerged that could further support diagnosis based on histopathological patterns. In the present work, formalin-fixed paraffin-embedded (FFPE) tissue specimens from normal (non-neoplastic) thyroid (normal controls (NCs)), benign tumors (follicular thyroid adenomas (FTAs)), and some common types of well-differentiated thyroid carcinoma (follicular thyroid carcinomas (FTCs), conventional or classical papillary thyroid carcinomas (CV-PTCs), and the follicular variant of papillary thyroid carcinomas (FV-PTCs)) were analyzed. For the first time, FFPE thyroid samples were deparaffinized using an easy, fast, and non-toxic method. Protein extracts from thyroid tissue samples were analyzed using a nanoparticle-assisted proteomics approach combined with shotgun LC-MS/MS. The differentially regulated proteins found to be specific for the FTA, FTC, CV-PTC, and FV-PTC subtypes were analyzed with the bioinformatic tools STRING and PANTHER showing a profile of proteins implicated in the thyroid cancer metabolic reprogramming, cancer progression, and metastasis. These proteins represent a new source of potential molecular targets related to thyroid tumors

Evaluation of Lu-177-Dotatate treatment in patients with metastatic neuroendocrine tumors and prognostic factors

BACKGROUND: (177)Lu peptide receptor radionuclide therapy (PRRT) is a recently approved therapy in Spain that has been demonstrated to be a well-tolerated therapy for positive somatostatin receptor advanced gastroenteropancreatic neuroendocrine tumors. AIM: To determine the impact of PRRT on quality of life, radiologic and metabolic response, overall survival, prognostic factors and toxicity. METHODS: Thirty-six patients treated with (177)Lu-PRRT from 2016 to 2019 were included. The most frequent location of the primary tumor was the gastrointestinal tract (52.8%), pancreas (27.8%), and nongastropancreatic neuroendocrine tumor (11.1%). The liver was the most common site of metastasis (91.7%), followed by distant nodes (50.0%), bone (27.8%), peritoneum (25.0%) and lung (11.1%). Toxicity was evaluated after the administration of each dose. Treatment efficacy was evaluated by two parameters: stable disease and disease progression in response evaluation criteria in solid tumors 1.1 criterion and prognostic factors were tested. RESULTS: From 36 patients, 55.6% were men, with a median age of 61.1 +/- 11.8 years. Regarding previous treatments, 55.6% of patients underwent surgery of the primary tumor, 100% of patients were treated with long-acting somatostatin analogues, 66.7% of patients were treated with everolimus, 27.8% of patients were treated with tyrosine kinase inhibitor, and 27.8% of patients were treated with interferon. One patient received radioembolization, three patients received chemoembolization, six patients received chemotherapy. Hematological toxicity was registered in 14 patients (G1-G2: 55.5% and G3: 3.1%). Other events presented were intestinal suboclusion in 4 cases, cholestasis in 2 cases and carcinoid crisis in 1 case. The median follow-up time was 3 years. Currently, 24 patients completed treatment. Nineteen are alive with stable disease, two have disease progression, eight have died, and nine are still receiving treatment. The median overall survival was 12.5 mo (95% confidence interval range: 9.8-15.2), being inversely proportional to toxicity in previous treatments (P < 0.02), tumor grade (P < 0.01) and the presence of bone lesions (P = 0.009) and directly proportional with matching lesion findings between Octreoscan and computed tomography pre-PRRT (P < 0.01), , primary tumor surgery (P = 0.03) and metastasis surgery (P = 0.045). In a multivariate Cox regression analysis, a high Ki67 index (P = 0.003), a mismatch in the lesion findings between Octreoscan and computed tomography pre-PRRT (P < 0.01) and a preceding toxicity in previous treatments (P < 0.05) were risk factors to overall survival. CONCLUSION: Overall survival was inversely proportional to previous toxicity, tumor grade and the presence of bone metastasis and directly proportional to matching lesion findings between Octreoscan and computed tomography pre-PRRT and primary tumor and metastasis surgery

LAT-1 and GLUT-1 Carrier Expression and Its Prognostic Value in Gastroenteropancreatic Neuroendocrine Tumors

Cancer cells develop mechanisms that increase nutrient uptake, including key nutrient carriers, such as amino acid transporter 1 (LAT-1) and glucose transporter 1 (GLUT-1), regulated by the oxygen-sensing Von Hippel Lindau-hypoxia-inducible factor (VHL-HIF) transcriptional pathway. We aimed to analyze these metabolic players in gastroenteropancreatic neuroendocrine tumors (GEP-NET) and correlate them with tumor malignancy and progression. LAT-1, GLUT-1, and pVHL expression was analyzed in 116 GEP-NETs and 48 peritumoral tissue samples by immunohistochemistry. LAT-1 was stably silenced using specific shRNA in the human NET BON cell line. LAT-1 expression was significantly increased in tumor tissue compared to non-tumor tissue in both gastrointestinal (67% vs. 44%) and pancreatic NETs (54% vs. 31%). Similarly, GLUT-1 was substantially elevated in gastrointestinal (74% vs. 19%) and pancreatic (58% vs. 4%) NETs. In contrast, pVHL expression was decreased (85% vs. 58%) in pancreatic NETs. Tumors with metastases at diagnosis displayed increased LAT-1 and GLUT-1 and decreased pVHL expression (p < 0.001). In accordance with these data, silencing LAT-1 curtailed cell proliferation in BON cells. These findings suggest that specific mechanisms that increase nutrient uptake, such as LAT-1 and GLUT-1, are increased in GEP-NETs, whereas pVHL is decreased. These markers might be related to the proliferation and metastatic capacity of these tumors

Should we suspect primary aldosteronism in patients with hypokalaemic rhabdomyolysis? A systematic review

Severe hypokalaemia causing rhabdomyolysis (RML) in primary aldosteronism (PA) is a rare entity, and only a few cases have been reported over the last four decades. This systematic review and case report aims to gather all published data regarding a hypokalaemic RML as presentation of PA in order to contribute to the early diagnosis of this extremely rare presentation. With the use of PubMed Central, EMBASE, and Google Scholar, a thorough internet-based search of the literature was conducted to identify articles and cases with RML secondary to hypokalaemia due to PA between June 1976 and July 2023. The case study concerns a 68-year-old male patient with hypokalaemic RML at presentation of PA. In the systematic review of the literature, 37 cases of RML secondary to hypokalaemia due to PA have been reported to date. In summary, the median age was 47.5 years, the male/female ratio was 17/21, all patients presented symptoms (weakness and/or myalgia), all the patients were hypertensive, and only four patients had complications with acute kidney injury (AKI). Although PA rarely presents with RML, it should be suspected when marked hypokalaemia and hypertension are also present. Early detection and management are essential to reduce the frequency of manifestations such as AKI

Humanized medium (h7H) allows long-term primary follicular thyroid cultures from human normal thyroid, benign neoplasm, and cancer

Mechanisms of thyroid physiology and cancer are principally studied in follicular cell lines. However, human thyroid cancer lines were found to be heavily contaminated by other sources, and only one supposedly normal-thyroid cell line, immortalized with SV40 antigen, is available. In primary culture, human follicular cultures lose their phenotype after passage. We hypothesized that the loss of the thyroid phenotype could be related to culture conditions in which human cells are grown in medium optimized for rodent culture, including hormones with marked differences in its affinity for the relevant rodent/human receptor.|The objective of the study was to define conditions that allow the proliferation of primary human follicular thyrocytes for many passages without losing phenotype.|Concentrations of hormones, transferrin, iodine, oligoelements, antioxidants, metabolites, and ethanol were adjusted within normal homeostatic human serum ranges. Single cultures were identified by short tandem repeats. Human-rodent interspecies contamination was assessed.|We defined an humanized 7 homeostatic additives medium enabling growth of human thyroid cultures for more than 20 passages maintaining thyrocyte phenotype. Thyrocytes proliferated and were grouped as follicle-like structures; expressed Na+/I- symporter, pendrin, cytokeratins, thyroglobulin, and thyroperoxidase showed iodine-uptake and secreted thyroglobulin and free T3. Using these conditions, we generated a bank of thyroid tumors in culture from normal thyroids, Grave's hyperplasias, benign neoplasms (goiter, adenomas), and carcinomas.|Using appropriate culture conditions is essential for phenotype maintenance in human thyrocytes. The bank of thyroid tumors in culture generated under humanized humanized 7 homeostatic additives culture conditions will provide a much-needed tool to compare similarly growing cells from normal vs pathological origins and thus to elucidate the molecular basis of thyroid disease.Ministerio de Ciencia e InnovaciónInstituto de Salud Carlos IIIXunta de GaliciaFondo Social Europeo of the European Communit