Single-cell mtDNA heteroplasmy in colorectal cancer.

Human mitochondria can be genetically distinct within the same individual, a phenomenon known as heteroplasmy. In cancer, this phenomenon seems exacerbated, and most mitochondrial mutations seem to be heteroplasmic. How this genetic variation is arranged within and among normal and tumor cells is not well understood. To address this question, here we sequenced single-cell mitochondrial genomes from multiple normal and tumoral locations in four colorectal cancer patients. Our results suggest that single cells, both normal and tumoral, can carry various mitochondrial haplotypes. Remarkably, this intra-cell heteroplasmy can arise before tumor development and be maintained afterward in specific tumoral cell subpopulations. At least in the colorectal patients studied here, the somatic mutations in the single-cells do not seem to have a prominent role in tumorigenesis.

Germline Mutations in PALB2, BRCA1, and RAD51C, Which Regulate DNA Recombination Repair, in Patients With Gastric Cancer.

Up to 10% of cases of gastric cancer are familial, but so far, only mutations in CDH1 have been associated with gastric cancer risk. To identify genetic variants that affect risk for gastric cancer, we collected blood samples from 28 patients with hereditary diffuse gastric cancer (HDGC) not associated with mutations in CDH1 and performed whole-exome sequence analysis. We then analyzed sequences of candidate genes in 333 independent HDGC and non-HDGC cases. We identified 11 cases with mutations in PALB2, BRCA1, or RAD51C genes, which regulate homologous DNA recombination. We found these mutations in 2 of 31 patients with HDGC (6.5%) and 9 of 331 patients with sporadic gastric cancer (2.8%). Most of these mutations had been previously associated with other types of tumors and partially co-segregated with gastric cancer in our study. Tumors that developed in patients with these mutations had a mutation signature associated with somatic homologous recombination deficiency. Our findings indicate that defects in homologous recombination increase risk for gastric cancer.

Cribriform-morular variant of thyroid carcinoma: a neoplasm with distinctive phenotype associated with the activation of the WNT/ß-catenin pathway.

Cribriform-morular variant of thyroid carcinoma is classically associated with familial adenomatous polyposis but, it can also occur as a sporadic neoplasm. This neoplasm is much more frequently observed in women than in men (ratio of 61:1). In familial adenomatous polyposis patients, tumors are generally multifocal and/or bilateral (multinodular appearance), whereas in the sporadic cases tumors tend to occur as single nodules. The tumors are well delimited, and characteristically show a blending of follicular, cribriform, papillary, trabecular, solid, and morular patterns. Neoplastic cells are tall or cuboidal with the occasional nuclear features of classic papillary thyroid carcinoma. The morules include cells with peculiar nuclear clearing and show positivity for CDX2 and CD10. Angioinvasion and capsular invasion have been described in about 30 and 40% of cases, respectively, with lymph node metastases in less than 10% of patients and distant metastases in 6%. Although this tumor has good prognosis, neuroendocrine and/or poor differentiation have been associated with aggressive behavior. Tumor cells can be focally positive or negative for thyroglobulin, but are always positive for TTF-1, estrogen and progesterone receptors, and negative for calcitonin and cytokeratin 20. Nuclear and cytoplasmic staining for ß-catenin is the hallmark of this tumor type; this feature plays a role in fine needle aspiration biopsy. Cribriform-morular variant of thyroid carcinoma has a peculiar endodermal (intestinal-like) type phenotype, activation of the WNT/ß-catenin signaling pathway, and belongs to the non-BRAF-non-RAS subtype of the molecular classification of thyroid tumors. Elevated expression of estrogen and progesterone receptors and activation of the WNT/ß-catenin pathway may prove useful as putative therapeutic targets in cases that do not respond to conventional therapy. Clinicians should be alerted to the possibility of familial adenomatous polyposis when a diagnosis of cribriform-morular variant of thyroid carcinoma is made. Instead of being considered as a variant of papillary thyroid carcinoma its designation as cribriform-morular thyroid carcinoma seems more appropriate.

Thyroid cancer GWAS identifies 10q26.12 and 6q14.1 as novel susceptibility loci and reveals genetic heterogeneity among populations.

Thyroid cancer is the most heritable cancer of all those not displaying typical Mendelian inheritance. However, most of the genetic factors that would explain the high heritability remain unknown. Our aim was to identify additional common genetic variants associated with susceptibility to this disease. In order to do so, we performed a genome-wide association study in a series of 398 cases and 502 controls from Spain, followed by a replication in four well-defined Southern European case-control collections contributing a total of 1,422 cases and 1,908 controls. The association between the variation at the 9q22 locus near FOXE1 and thyroid cancer risk was consistent across all series, with several SNPs identified (rs7028661: OR = 1.64, p = 1.0 × 10(-22) , rs7037324: OR = 1.54, p = 1.2 × 10(-17) ). Moreover, the rare alleles of three SNPs (rs2997312, rs10788123 and rs1254167) at 10q26.12 showed suggestive evidence of association with higher risk of the disease (OR = 1.35, p = 1.2 × 10(-04) , OR = 1.26, p = 5.2 × 10(-04) and OR = 1.38, p = 5.9 × 10(-05) , respectively). Finally, the rare allele of rs4075570 at 6q14.1 conferred protection in the series studied (OR = 0.82, p = 2.0 × 10(-04) ). This study suggests that heterogeneity in genetic susceptibility between populations is a key feature to take into account when exploring genetic risk factors related to this disease.

dsRNAi-mediated silencing of PIAS2beta specifically kills anaplastic carcinomas by mitotic catastrophe.

The E3 SUMO ligase PIAS2 is expressed at high levels in differentiated papillary thyroid carcinomas but at low levels in anaplastic thyroid carcinomas (ATC), an undifferentiated cancer with high mortality. We show here that depletion of the PIAS2 beta isoform with a transcribed double-stranded RNA-directed RNA interference (PIAS2b-dsRNAi) specifically inhibits growth of ATC cell lines and patient primary cultures in vitro and of orthotopic patient-derived xenografts (oPDX) in vivo. Critically, PIAS2b-dsRNAi does not affect growth of normal or non-anaplastic thyroid tumor cultures (differentiated carcinoma, benign lesions) or cell lines. PIAS2b-dsRNAi also has an anti-cancer effect on other anaplastic human cancers (pancreas, lung, and gastric). Mechanistically, PIAS2b is required for proper mitotic spindle and centrosome assembly, and it is a dosage-sensitive protein in ATC. PIAS2b depletion promotes mitotic catastrophe at prophase. High-throughput proteomics reveals the proteasome (PSMC5) and spindle cytoskeleton (TUBB3) to be direct targets of PIAS2b SUMOylation at mitotic initiation. These results identify PIAS2b-dsRNAi as a promising therapy for ATC and other aggressive anaplastic carcinomas.

Vaginal tubulovillous adenoma: a clinicopathologic and molecular study with review of the literature.

Vaginal villous or tubulovillous adenomas (TVA) are uncommon tumors histologically similar to their intestinal counterparts. After reviewing the literature, we report the eighth case of TVA, which presented as a polypoid tumor in the vagina, at suburethral level, in a 19-yr-old woman with Arnold-Chiari type II malformation and a myelomeningocele at birth. The tumor consisted of long villi lined by columnar cells with brush borders, pseudostratified nuclei, and foci of high-grade atypia. Immunohistochemistry was positive for cytokeratin 7, estrogen and progesterone receptors, CA19.9, p16, p53, and Ki-67 (53%), with a normal membranous pattern for ß-catenin, but negative for cytokeratin 20, CDX2, carcinoembryonic antigen, chromogranin A, and synaptophysin. Neither human papillomavirus nor mutations in the K-RAS, BRAF, or LKB1/STK11 genes were detected. Although a rare neoplasm, awareness of this tumor is important as it must be distinguished from colonic adenocarcinoma or other malignant or benign conditions. The existence of 2 previously reported malignant cases merging with TVAs, and the presence of foci of high-grade dysplasia (p53-positive) in the present case, support TVA as a premalignant lesion.

Madelung's disease: comorbidities, fatty mass distribution, and response to treatment of 22 patients.

BACKGROUND: Madelung's disease (MD) is an uncommon pathology characterized by the presence of multiple masses of unencapsulated adipose tissue that is symmetrically distributed. The aim of this study was to investigate clinical and epidemiological features of patients diagnosed with MD in our department. Associated diseases and evolution after treatment were also investigated. METHODS: We reviewed the clinical histories of 22 patients diagnosed with MD from 1990 to 2010 and obtained their epidemiological and clinical characteristics. RESULTS: We found 21 patients with MD type 1 and one patient with MD type 2 according to Enzi's classification. All patients were male, 95.5% with high alcohol intake, and 59.1% with some hepatic disease. No family antecedents were significant; 40.9% had dyslipidemia, 22.7% arterial hypertension, 22.7% chronic obstructive pulmonary disease (COPD), 13.6% hyperuricemia, 9.1% hypothyroidism, 4.5% diabetes mellitus type 2, and 4.5% carbohydrate intolerance; 40.9% had a body mass index>30, and 27.3% presented gynecomastia/lipomastia. The region most frequently affected by fatty deposits was the neck. CONCLUSIONS: Madelung's disease affects mainly alcoholic males in their fourth decade of life. Hepatic diseases appear in most patients. Also associated with MD are high lipid blood levels, arterial hypertension, COPD, hyperuricemia, and obesity. MD type 1 is the most frequent phenotype and the neck the most common location for fatty masses. Recurrence after surgery, in the same location or different locations, is a frequent event, even in patients who later abstain from alcohol intake. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors at www.springer.com/00266 .

Cribriform morular thyroid carcinoma: a case report with pathological, immunohistochemical, and molecular findings suggesting an origin from follicular cells (or their endodermal precursors).

Cribriform morular thyroid carcinoma (CMTC) is a rare malignant thyroid tumor with a peculiar growth pattern secondary to permanent activation of the WNT/ß-catenin pathway. CMTC may be associated with familial adenomatous polyposis or sporadic; it shares morphological features with papillary thyroid carcinoma (PTC) and was considered a variant of PTC in the 2017 WHO classification of tumors of endocrine organs. The new 5th edition of the WHO classification of endocrine and neuroendocrine tumors considered CMTC an independent thyroid neoplasm of uncertain histogenesis. A thymic/ultimobranchial pouch-related differentiation in CMTC has been recently postulated. We, however, have used the pathological and immunohistochemical features of this case of CMTC with 2 novel oncogenic somatic variants (c.3428_3429insA, p.(Tyr1143Ter) and c.3565del, p. (Ser1189Hisfs*76) of the APC gene to propose an origin from follicular cells (or their endodermal precursors). As usual in CMTC, the morular component of this tumor was positive for CDX2. Given the fact that WNT/ß-catenin signaling, through CDX2, activates large intestine and small intestine gene expression, we postulate that in CMTC, the tumor cells have their terminal differentiation blocked, thus showing a peculiar primitive endodermal (intestinal-like) phenotype negative for sodium-iodide symporter, thyroperoxidase, and thyroglobulin. Establishing the histogenesis of CMTC is very relevant for the development of appropriate therapies of redifferentiation, particularly in patients where the tumor cannot be controlled by surgery.

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.

Adrenal Medullary Hyperplasia: An Under the Radar Cause of Endocrine Hypertension.

Adrenal medullary hyperplasia is a cause of increased secretion of catecholamines by the adrenal gland that is rarely considered among the differential diagnoses of endocrine hypertension. We report the case of a 48-year-old Hispanic woman who presented for evaluation of resistant hypertension with several episodes of hypertensive crisis. The clinical presentation, biochemical results, and abdominal computed tomography scan suggested the possibility of a pheochromocytoma; however, an iodine-123-meta-iodobenzylguanidine (123I-MIBG) uptake study combined with single-photon emission computed tomography (SPECT) and computed tomography (CT) scan showed diffusely increased metabolic activity in both adrenal glands. The patient underwent left adrenalectomy, and the pathology study revealed the presence of adrenal medullary hyperplasia. After surgery, blood pressure control was achieved with one antihypertensive drug, and the patient did not have recurrent hypertensive crisis. Relevant findings obtained from a whole genomic sequence done on a whole blood DNA sample from the patient are discussed.

Susceptibility Genes and Chromosomal Regions Associated With Non-Syndromic Familial Non-Medullary Thyroid Carcinoma: Some Pathogenetic and Diagnostic Keys.

Thyroid cancer is the malignant tumor that is increasing most rapidly in the world, mainly at the expense of sporadic papillary thyroid carcinoma. The somatic alterations involved in the pathogenesis of sporadic follicular cell derived tumors are well recognized, while the predisposing alterations implicated in hereditary follicular tumors are less well known. Since the genetic background of syndromic familial non-medullary carcinoma has been well established, here we review the pathogenesis of non-syndromic familial non-medullary carcinoma emphasizing those aspects that may be useful in clinical and pathological diagnosis. Non-syndromic familial non-medullary carcinoma has a complex and heterogeneous genetic basis involving several genes and loci with a monogenic or polygenic inheritance model. Most cases are papillary thyroid carcinoma (classic and follicular variant), usually accompanied by benign thyroid nodules (follicular thyroid adenoma and/or multinodular goiter). The possible diagnostic and prognostic usefulness of the changes in the expression and/or translocation of various proteins secondary to several mutations reported in this setting requires further confirmation. Given that non-syndromic familial non-medullary carcinoma and sporadic non-medullary thyroid carcinoma share the same morphology and somatic mutations, the same targeted therapies could be used at present, if necessary, until more specific targeted treatments become available.

Inherited Follicular Epithelial-Derived Thyroid Carcinomas: From Molecular Biology to Histological Correlates.

Cancer derived from thyroid follicular epithelial cells is common; it represents the most common endocrine malignancy. The molecular features of sporadic tumors have been clarified in the past decade. However the incidence of familial disease has not been emphasized and is often overlooked in routine practice. A careful clinical documentation of family history or familial syndromes that can be associated with thyroid disease can help identify germline susceptibility-driven thyroid neoplasia. In this review, we summarize a large body of information about both syndromic and non-syndromic familial thyroid carcinomas. A significant number of patients with inherited non-medullary thyroid carcinomas manifest disease that appears to be sporadic disease even in some syndromic cases. The cytomorphology of the tumor(s), molecular immunohistochemistry, the findings in the non-tumorous thyroid parenchyma and other associated lesions may provide insight into the underlying syndromic disorder. However, the increasing evidence of familial predisposition to non-syndromic thyroid cancers is raising questions about the importance of genetics and epigenetics. What appears to be "sporadic" is becoming less often truly so and more often an opportunity to identify and understand novel genetic variants that underlie tumorigenesis. Pathologists must be aware of the unusual morphologic features that should prompt germline screening. Therefore, recognition of harbingers of specific germline susceptibility syndromes can assist in providing information to facilitate early detection to prevent aggressive disease.

Pathological thyroid findings in amiodarone-induced thyrotoxicosis.

Amiodarone (AMD) is a class III antiarrhythmic drug whose chronic or high dosage administration alters the tests of thyroid function. AMD is also associated with hypothyroidism or thyrotoxicosis. Total thyroidectomy is an efficient treatment of AMD-induced thyrotoxicosis in cases resistant to medical therapy, worsening of cardiac function and/or severe thyrotoxicosis. Although AMD is a widely used drug, its pathological consequences are not well known. We describe the pathological findings in the thyroid gland of a patient who underwent total thyroidectomy due to AMD-induced thyrotoxicosis. The surgical specimen was macroscopically normal, but histologically showed multiple follicles totally or partially invaded by clear vacuolated (foamy) histiocytes, sometimes multinucleated. Loss of thyrocytes, breaks in the follicular basal membrane and stromal fibrosis could also be appreciated but no lymphocytic infiltrates were found. An awareness of these histopathological features is particularly important for surgical pathologists, especially as there are very few published reports describing these alterations.

Molecular Pathology of Non-familial Follicular Epithelial-Derived Thyroid Cancer in Adults: From RAS/BRAF-like Tumor Designations to Molecular Risk Stratification.

This review addresses the impact of molecular alterations on the diagnosis and prognosis of differentiated thyroid carcinoma (DTC), including papillary, follicular, and well-differentiated carcinoma NOS, as well as oncocytic neoplasms. The molecular characterization of DTC is based upon the well-established dichotomy of BRAF-like and RAS-like designations, together with a remaining third group, less homogeneous, composed of non-BRAF-/non-RAS-like tumors. The role of BRAF V600E mutation in risk stratification is discussed in the clinico-pathological context, namely, staging and invasive features of classic papillary thyroid carcinoma (PTC) and histopathological variants carrying an excellent prognosis (microPTC) or a guarded prognosis, including the aggressive variants tall cell and hobnail cell PTCs. In follicular patterned tumors, namely, follicular thyroid carcinoma (FTC), with or without oncocytic features, the most prevalent molecular alteration are RAS mutations that do not carry prognostic significance. The only genetic alteration that has been proven to play a role in risk stratification of PTC and FTC is TERT promoter (TERTp) mutation.

PD-L1 expression and immune cells in anaplastic carcinoma and poorly differentiated carcinoma of the human thyroid gland: A retrospective study.

Anaplastic thyroid carcinoma (ATC) and poorly differentiated thyroid carcinoma (PDTC) have limited treatment options, and immune profiling may help select patients for immunotherapy. The prevalence and relevance of programmed death-1 ligand (PD-L1) expression and the presence of immune cells in ATC and PDTC has not yet been well established. The present study investigated PD-L1 expression (clone 22C3) and cells in the tumor microenvironment (TME), including tumor-infiltrating lymphocytes (TILs), tumor-associated macrophages (TAMs) and dendritic cells, in whole tissue sections of 15 cases of ATC and 13 cases of PDTC. Immunohistochemical PD-L1 expression using a tumor proportion score (TPS) with a 1% cut-off was detected in 9/15 (60%) of ATC cases and 1/13 (7.7%) of PDTC cases (P=0.006). PD-L1 expression in TILs was limited to the ATC group (73.3 vs. 0% in ATC and PDTC, respectively). In the ATC group, the TPS for tumor positive PD-L1 expression revealed a non-significant trend towards worse survival, but no difference was observed when investigating PD-L1 expression in TILs and TAMs. In addition to increased PD-L1 expression, all ATC cases exhibited significantly increased CD3+ and CD8+ T cells, CD68+ and CD163+ macrophages, and S100+ dendritic cells compared with the PDTC cases. Loss of mutL homolog 1 and PMS1 homolog 2 expression was observed in one ATC case with the highest PD-L1 expression, as well as in the only PDTC case positive for PD-L1. Notably, the latter was the only PDTC case exhibiting positivity for p53 and a cellular microenvironment similar to ATC. The current results indicated that PD-L1 expression was frequent in ATC, but rare in PDTC. In addition to PD-L1, the present study suggested that microsatellite instability may serve a role in both the TME and the identification of immunotherapy candidates among patients with PDTC.

Protein Extraction From FFPE Kidney Tissue Samples: A Review of the Literature and Characterization of Techniques.

Most tissue biopsies from patients in hospital environments are formalin-fixed and paraffin-embedded (FFPE) for long-term storage. This fixation process produces a modification in the proteins called "crosslinks", which improves protein stability necessary for their conservation. Currently, these samples are mainly used in clinical practice for performing immunohistochemical analysis, since these modifications do not suppose a drawback for this technique; however, crosslinks difficult the protein extraction process. Accordingly, these modifications make the development of a good protein extraction protocol necessary. Due to the specific characteristics of each tissue, the same extraction buffers or deparaffinization protocols are not equally effective in all cases. Therefore, it is necessary to obtain a specific protocol for each tissue. The present work aims to establish a deparaffinization and protein extraction protocol from FFPE kidney samples to obtain protein enough of high quality for the subsequent proteomic analysis. Different deparaffination, protocols and protein extraction buffers will be tested in FFPE kidney samples. The optimized conditions will be applied in the identification by LC-MS/MS analysis of proteins extracted from 5, 10, and 15 glomeruli obtained through the microdissection of FFPE renal samples.

Rate of non-invasive follicular thyroid neoplasms with papillary-like nuclear features depends on pathologist's criteria: a multicentre retrospective Southern European study with prolonged follow-up.

PURPOSE: To determine the rate of non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) in a multi-institutional series from the Iberian Peninsula and describing this NIFTP cohort. METHODS: Retrospective study of papillary thyroid carcinoma (PTC) or well-differentiated tumours of uncertain malignant potential (WDT-UMP) diagnosed between 2005 and 2015 and measuring ≥5 mm in adult patients from 17 hospitals. Pathological reports were reviewed to determine the cases that fulfil the original criteria of NIFTP and histology was reassessed. Rates were correlated with the number of PTC and its follicular variant (FVPTC) of each institution. Demographic data, histology, management, and follow-up of the reclassified NIFTP cohort were recorded. RESULTS: A total of 182 cases with NIFTP criteria were identified: 174/3372 PTC (rate: 5.2%; range: 0-12.1%) and 8/19 WDT-UMP (42.1%). NIFTP rate showed linear correlation with total PTC (p: 0.03) and FVPTC (p: 0.007) identified at each centre. Ultrasound findings were non-suspicious in 60.1%. Fine-needle cytology or core biopsy diagnoses were undetermined in 49.7%. Most patients were treated with total thyroidectomy. No case had nodal disease. Among patients with total thyroidectomy, 89.7% had an excellent response evaluated 1 year after surgery. There were no structural persistence or relapses. Five patients showed residual thyroglobulin after 90 months of mean follow-up. CONCLUSIONS: NIFTP rate is low but highly variable in neighbouring institutions of the Iberian Peninsula. This study suggests pathologist's interpretation of nuclear alterations as the main cause of these differences. Patients disclosed an excellent outcome, even without using the strictest criteria.

Expression of exogenous proteins and short hairpin RNAs in human primary thyrocytes.

Recently, it has been shown that commercial human thyroid lines were in fact derived from colon, mammary carcinoma, or melanoma. Others have demonstrated the absence of a common pattern of gene expression between available thyroid cancer cell lines and tumors from patients. Thus, it is important to use several primary cells with a common pathological origin to achieve reproducible results, and it is necessary to find common methods for manipulation of protein expression in such various cultures. We have standardized a transfection method for efficient expression of exogenous proteins in human primary thyroid cultures. We compared lipid-based techniques with three electroporation systems (Electroporator PulseAgile [PA]-4000, Microporator MP-100, and Nucleofector II). Nucleofection was unquestionably the most efficient even for promoter regulation studies, and it was effective in cultures from different origins as normal thyroid, papillary carcinoma, or lymphoid node metastasis. We also standardized, through lentiviral infection, the short hairpin RNA downregulation of protein expression generating human thyrocytes with low levels of p27KIP1 as a model system.

Pitfalls in Challenging Thyroid Tumors: Emphasis on Differential Diagnosis and Ancillary Biomarkers.

Thyroid pathology encompasses a heterogenous group of clinicopathological entities including rare and diagnostically challenging neoplasms. The review is focused on morphological, immunohistochemical, and molecular features of rare thyroid neoplasms that can pose diagnostic problems. The tumors are organized based on growth patterns including thyroid neoplasms with predominantly papillary, follicular, solid, and spindle cell growth pattern, as well as neoplasms with distinct cytological characteristics. A special section is also dedicated to rare thyroid tumors with peculiar patterns including thyroid carcinoma with Ewing family tumor elements and intrathyroidal thymic-related neoplasms.

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.