NKX3.1 Expression in Non-Prostatic Tumors and Characterizing its Expression in Esophageal/Gastroesophageal Adenocarcinoma.

The NKX3.1 immunohistochemical stain is widely recognized as a highly sensitive and specific marker for prostate adenocarcinoma. Nevertheless, its expression has been documented in various nonprostatic tissues and malignancies. This review aims to provide an overview of NKX3.1 expression in diverse tumor types, with a specific focus on its aberrant expression in esophageal/gastroesophageal adenocarcinoma (E/GE-ADC). In our investigation, we explored the expression of NKX3.1 in a series of E/GE-ADC to shed light on its prevalence in this tumor category. A total of 50 samples, comprising primary and metastatic E/GE-ADC specimens from 34 patients, were subjected to immunohistochemical analysis. Stained sections were scored based on the intensity and distribution-categorized as negative, weak, moderate, or strong in either a focal or diffuse pattern. Strong staining corresponds to the intensity observed in normal prostate controls, while focal and diffuse staining denote <50% and ≥50% of tumor nuclei staining positive, respectively. Our semiquantitative scoring revealed that 6 (12%) of the primary and metastatic E/GE-ADC specimens exhibited variable positivity for NKX3.1. This finding suggests that E/GE-ADC can sporadically stain positive for NKX3.1, introducing potential challenges in definitively determining the primary site of origin in certain clinical scenarios. Along with a literature review of NKX3.1 expression in other tumor types, our study provides additional important information about the extent to which this immunostain can be seen in E/GE-ADCs, which, to our knowledge, has not been reported.

Proximity ligation assay to detect DUX4 protein in FSHD1 muscle: a pilot study.

OBJECTIVE: Aberrant expression in skeletal muscle of DUX4, a double homeobox transcription factor, underlies pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). Although previous studies of FSHD muscle biopsies detected mRNAs encoding DUX4 and its target genes, no studies had reported detection of DUX4 protein. Our objective was to develop a proximity ligation assay (PLA) for DUX4 and to determine if this assay could detect DUX4 protein in FSHD muscle sections. RESULTS: We developed a PLA protocol using two DUX4 antibodies previously reported by Stephen Tapscott's group: P2G4, a mouse mAb specific for an epitope in the N-terminal region, and E5-5, a rabbit mAb specific for an epitope in the C-terminal region, in combination with commercial PLA secondary reagents. We validated the DUX4 PLA using cultured human myogenic cells in which DUX4 was ectopically expressed in a small fraction of nuclei. Using this two primary mAb PLA on an FSHD1 biceps biopsy, we observed nuclei with apparent DUX4 PLA signals associated with a small subset of myofibers (~ 0.05-0.1%). Though a limited pilot study, these results suggest that the two primary mAb PLA protocol could be useful for detecting DUX4 protein in FSHD muscle biopsies.

Interaction of human CRX and NRL in live HEK293T cells measured using fluorescence resonance energy transfer (FRET).

CRX and NRL are retina-specific transcription factors that control rod photoreceptor differentiation and synergistically activate rod phototransduction gene expression. Previous experiments showed they interact in vitro and in yeast two-hybrid assays. Here, we examined CRX-NRL interaction in live HEK293T cells using two fluorescence resonance energy transfer (FRET) approaches: confocal microscopy and flow cytometry (FC-FRET). FC-FRET can provide measurements from many cells having wide donor-acceptor expression ranges. FRET efficiencies were calibrated with a series of donor (EGFP)-acceptor (mCherry) fusion proteins separated with linkers between 6-45 amino acids. CRX and NRL were fused at either terminus with EGFP or mCherry to create fluorescent proteins, and all combinations were tested in transiently transfected cells. FRET signals between CRX or NRL homo-pairs were highest with both fluorophores fused to the DNA binding domains (DBD), lower with both fused to the activation domains (AD), and not significant when fused on opposite termini. NRL had stronger FRET signals than CRX. A significant FRET signal between CRX and NRL hetero-pairs was detected when donor was fused to the CRX DNA binding domain and the acceptor fused to the NRL activation domain. FRET signals increased with CRX or NRL expression levels at a rate much higher than expected for collisional FRET alone. Together, our results show the formation of CRX-NRL complexes in live HEK293T cells that are close enough for FRET.

Does PAX7 and NKX2.2 immunoreactivity in Ewing sarcoma have prognostic significance?

Ewing sarcoma (ES) is an aggressive neoplasm with variable morphology. It has no specific immunoprofile or molecular signature. Neither CD99, NKX2.2 nor PAX7 immunoreactivity alone is completely specific, although diagnostic specificity improves when combined. The purpose of the present study was to investigate the immunohistochemical (IHC) expression of PAX7 in a large series of genetically confirmed ES. Existing results for CD99 and NKX2.2 immunoexpression, morphological findings and molecular studies (fusion gene subtypes) were retrieved from a previous study. Survival analyses were performed in cases with available clinical follow-up. PAX7 was positive in 95.5% of ES with diffuse staining (> 50%) in all positive cases and moderate or strong intensity for most cases. Nineteen ES displayed both PAX7 and CD99 immunoreactivity but lacked NKX2.2 immunoexpression. No relationships could be found between PAX7 expression and the histological types or ES gene fusion subtypes. Univariant/multivariate analysis showed that lack of PAX7 and/or NKX2.2 immunoexpression constitute independent poor prognostic factors for progression free survival (PFS) and overall survival (OS). In conclusion, IHC for CD99, NKX2.2, and PAX7 may be useful in daily practice for ES diagnosis, particularly in hospitals lacking facilities for molecular studies. In addition, the combination of strong CD99 membranous positivity and nuclear PAX7 and NKX2.2 immunoreactivity seems to be very reliable for ES diagnosis when supported by a corroborating histomorphologic and clinical picture. Although PAX7 is not entirely specific for ES, it seems to have a more extensive and strong nuclear immunoreactivity than NKX2.2 expression, even in tumors with decalcification artifact. Considering the prognostically significant data herein reported, we strongly recommend validation in prospective ES series that include localized and disseminated tumors.

Metastatic Patterns of Duodenopancreatic Neuroendocrine Tumors in Patients With Multiple Endocrine Neoplasia Type 1.

Patients with multiple endocrine neoplasia 1 syndrome (MEN1) often develop multifocal duodenopancreatic neuroendocrine tumors (dpNETs). Nonfunctional pancreatic neuroendocrine tumors (PanNETs) and duodenal gastrinomas are the most frequent origins of metastasis. Current guidelines recommend surgery based on tumor functionality, size ≥2 cm, grade or presence of lymph node metastases. However, in case of multiple primary tumors it is often unknown which specific tumor metastasized. This study aims to unravel the relationship between primary dpNETs and metastases in patients with MEN1 by studying endocrine differentiation. First, it was shown that expression of the endocrine differentiation markers ARX and PDX1 was concordant in 18 unifocal sporadic neuroendocrine tumors (NETs) and matched metastases. Thereafter, ARX, PDX1, Ki67 and gastrin expression, and the presence of alternative lengthening of telomeres were determined in 137 microscopic and macroscopic dpNETs and 36 matched metastases in 10 patients with MEN1. ARX and PDX1 H-score clustering was performed to infer relatedness. For patients with multiple metastases, similar intrametastases transcription factor expression suggests that most metastases (29/32) originated from a single NET of origin, while few patients may have multiple metastatic primary NETs. In 6 patients with MEN1 and hypergastrinemia, periduodenopancreatic lymph node metastases expressed gastrin, and clustered with minute duodenal gastrinomas, not with larger PanNETs. PanNET metastases often clustered with high grade or alternative lengthening of telomeres-positive primary tumors. In conclusion, for patients with MEN1-related hypergastrinemia and PanNETs, a duodenal origin of periduodenopancreatic lymph node metastases should be considered, even when current conventional and functional imaging studies do not reveal duodenal tumors preoperatively.

Identification of early diagnostic biomarkers via WGCNA in gastric cancer.

BACKGROUND: Gastric cancer (GC) is the world's second-leading cause of cancer-related mortality, continuing to make it a serious healthcare concern. Even though the prevalence of GC reduces, the prognosis for GC patients remains poor in terms of a lack of reliable biomarkers to diagnose early GC and predict chemosensitivity and recurrence. METHODS AND MATERIAL: We integrated the gene expression patterns of gastric cancers from four RNAseq datasets (GSE113255, GSE142000, GSE118897, and GSE130823) from Gene Expression Omnibus (GEO) database to recognize differentially expressed genes (DEGs) between normal and GC samples. A gene co-expression network was built using weighted co-expression network analysis (WGCNA). Furthermore, RT-qPCR was performed to validate the in silico results. RESULTS: The red modules in GSE113255, Turquoise in GSE142000, Brown in GSE118897, and the green-yellow module in GSE130823 datasets were found to be highly correlated with the anatomical site of GC. ITGAX, CCL14, ADHFE1, and HOXB13) as the hub gene are differentially expressed in tumor and non-tumor gastric tissues in this study. RT-qPCR demonstrated a high level of the expression of this gene. CONCLUSION: The expression levels of ITGAX, CCL14, ADHFE1, and HOXB13 in GC tumor tissues are considerably greater than in adjacent normal tissues. Systems biology approaches identified that these genes could be possible GC marker genes, providing ideas for other experimental studies in the future.

PHOX2B as a Reliable Marker for Neuroblastoma in Tissue and Cytology Specimens.

To investigate the diagnostic utility of immunohistochemistry for paired-like homeobox 2B (PHOX2B) expression in neuroblastomas (NBs) and tumors that mimic them, tissue samples (n = 229) from 157 cases of NB, 210 central nervous system tumors, and 170 extracranial non-NB solid tumors (n = 170) were immunostained for PHOX2B. Additionally, PHOX2B expression in 67 body fluid cytology specimens was analyzed. In tissue specimens, PHOX2B expression was positive in NBs, pheochromocytomas, and paragangliomas but negative in all of the other tumors evaluated. PHOX2B was detected by immunohistochemistry in 5 NB cytology specimens; all of the others were negative. These results suggest that PHOX2B may be a sensitive and specific immunohistochemical marker for the pathological diagnosis and differential diagnosis of NB in both tissue and cytology specimens.

HOXD10 expression as a prognostic factor for hepatocellular carcinoma treated with curative resection.

PURPOSE: HOXD10 downregulation resulting from epigenetic changesas well as its role as a tumor suppressor have been reported in several cancers including hepatocellular carcinomas (HCCs). However, the prognostic role of HOXD10 expression in HCC tissue samples has not been evaluated. METHODS: HOXD10 expression was investigated in 278 curatively resected HCC samples using immunohistochemistry and its effectiveness in predicting patient outcome was analyzed. RESULTS: Low expression of HOXD10 was observed in 82.7% of HCC samples, and this was associated with increased age, large tumor size and advanced stage.HOXD10 was an independent predictive factor for early tumor recurrence at less than 2 years. Patients with low HOXD10 expression showed shorter recurrence-free survival (RFS) (p=0.024) and disease-specific survival (DSS) (p=0.016) than those with high expression. Multivariate analysis confirmed that low HOXD10 expression was an independent predictor of shorter RFS (hazard ratio 1.873, p=0.006) and DSS (hazard ratio2.504, p=0.012) than high HOXD10 expression. CONCLUSIONS: The present study provides clinical evidence supporting the use of HOXD10 as a prognostic biomarker in curatively resected HCCs, and suggests that HOXD10 could also be a potential therapeutic target in HCC.

[Diagnostic Efficacy of SHOX2 Gene Hypermethylation for Lung Cancer: A Meta-Analysis].

BACKGROUND: Lung cancer is the most common malignant tumor in clinic. The prognosis of advanced patients is poor, and the 5-year survival rate is low. Therefore, early diagnosis becomes the key to improve the prognosis of patients. In recent years, with the development of molecular biology technology, aberrant modification of some driver genes, such as methylation, has become an important method for early diagnosis of lung cancer. The purpose of the present work was to quantitatively evaluate the diagnostic value of abnormal hypermethylation in short state homeobox 2 (SHOX2) promoter region in lung cancer by evidence-based medicine. METHODS: We searched MEDLINE, EMBASE, Ovid, Web of Science and CNKI for literatures related to the relationship between SHOX2 gene promoter hypermethylation and lung cancer. The data of SHOX2 promoter hymethylation in the original study were extracted. The diagnostic sensitivity, specificity and area under receiver operating characteristic (ROC) curve of SHOX2 promoter methylation were calculated. RESULTS: Finally, 13 publications were included in this meta-analysis, and due to significant statistical heterogeneity among the studies (P<0.05) the data was pooled by random effect model. The diagnostic sensitivity and specificity of SHOX2 promoter hypermethylation in the diagnosis of lung cancer were 0.75 (95%CI: 0.74-0.77) and 0.89 (95%CI: 0.88-0.91), respectively; The positive likelihood ratio value was 6.75 (4.56-9.99), and the negative predictive value was 0.36 (0.25-0.52); The diagnostic odds ratio was 23.16 (11.34-47.31), and the area under the ROC curve was 0.9. CONCLUSIONS: SHOX2 gene promoter hypermethylation is high in serum, broncholavage fluid and pleural effusion of lung cancer patients, which can be used as a biomarker for auxiliary diagnosis of lung cancer.

Expanding the Spectrum of EWSR1-NFATC2-rearranged Benign Tumors: A Common Genomic Abnormality in Vascular Malformation/Hemangioma and Simple Bone Cyst.

A simple bone cyst (SBC) is a cystic bone lesion predominantly affecting young males. The cyst is lined by a fibrous membrane and filled with serosanguinous fluid. EWSR1/FUS-NFATC2 rearrangements were recently identified in SBC. We here report exactly the same rearrangement in 3 lesions diagnosed as vascular malformations of 2 elderly patients. In total, through Archer FusionPlex, fluorescence in situ hybridization and/or reverse transcriptase-polymerase chain reaction the EWSR1-NFATC2 rearrangement was identified in 6 of 9 SBC, 3 of 12 benign vascular tumors, and none of 5 aneurysmal bone cyst lacking USP6 fusion. Using fluorescence in situ hybridization, it was apparent that amplification of the fusion, as seen in EWSR1-NFATC2 round cell sarcomas, was absent, and that in the vascular tumors the fusion was present both in the lining cells as well as in the surrounding spindle cells. Of note, not all of the spaces in the vascular malformations were lined by endothelial cells. Aggrecan was positive in all cases but was not specific. NKX2-2 and NKX3-1 staining were negative in all cases. Thus, even though the overlap between the 2 entities is limited to the presence of few thick-walled cysts lacking endothelial lining in the benign vascular malformations, the spectrum of benign tumors containing NFATC2 fusions should be expanded and contains not only SBC in the young, but also vascular malformation/hemangioma in elderly patients.

New lymphatic cell formation is associated with damaged brain tissue clearance after penetrating traumatic brain injury.

We characterized the tissue repair response after penetrating traumatic brain injury (pTBI) in this study. Seventy specific pathogen-free Kunming mice were randomly divided into the following groups: normal control, 1, 3, 7, 15, 21, and 30 days after pTBI. Hematoxylin and eosin (H&E) staining, immunohistochemistry, and immunofluorescence were performed to examine and monitor brain tissue morphology, and the distribution and expression of lymphatic-specific markers lymphatic vessel endothelial receptor-1 (LYVE-1), hematopoietic precursor cluster of differentiation 34 (CD34) antigen, and Prospero-related homeobox-1 (PROX1) protein. H&E staining revealed that damaged and necrotic tissues observed on day 1 at and around the injury site disappeared on day 7, and there was gradual shrinkage and disappearance of the lesion on day 30, suggesting a clearance mechanism. We explored the possibility of lymphangiogenesis causing this clearance as part of the post-injury response. Notably, expression of lymphangiogenesis markers LYVE-1, CD34, and PROX1 was detected in damaged mouse brain tissue but not in normal tissue. Moreover, new lymphatic cells and colocalization of LYVE-1/CD34 and LYVE-1/PROX1 were also observed. Our findings of the formation of new lymphatic cells following pTBI provide preliminary insights into a post-injury clearance mechanism in the brain. Although we showed that lymphatic cells are implicated in brain tissue repair, further research is required to clarify the origin of these cells.

Overexpression of Teashirt Homolog 2 suppresses cell proliferation and predicts the favorable survival of Lung Adenocarcinoma.

Background: Teashirt homolog 2 (TSHZ2) is essential for maintaining cellular homeostasis and regulating transcription on neoplasia development. However, the regulation of TSHZ2 in lung tumorigenesis and the underlying mechanisms remain unclear. Objective: To evaluate the relationship between TSHZ2 expression in patients' tumor tissue and prognosis in lung adenocarcinoma. Methods: TSHZ2 expression in different lung adenocarcinoma cell lines and human tissue were detected by Western blotting. The effect of TSHZ2 on cell proliferation, apoptosis and migration in lung adenocarcinoma cells was measured by CCK8, colony formation, flowcytometric analyses and wound-healing, respectively. TSHZ2 expression in different lung adenocarcinoma cell lines and human tissue from patients was detected using Western blotting and immunohistochemical staining. We also retrospectively analyzed 226 lung adenocarcinoma patients after surgical resection using immunohistochemical staining, and the association of TSHZ2 expression with the patient survival was evaluated. Results: TSHZ2 was lowly expressed in certain lung adenocarcinoma cell lines (PC9 and B203L), but other cells showed a high expression. Low expression of TSHZ2 was also observed in most lung adenocarcinoma tissues compared with adjacent tissues. Furthermore, we found that the overexpression of TSHZ2 plasmids led to the dramatic inhibition of cell proliferation, colony formation ability, migration and apoptosis induction in PC9 lung adenocarcinoma cells. In contrast, no obvious effect was found when the TSHZ2 expression was down-regulated by si-TSHZ2. An elevated TSHZ2 expression was observed in 155(68.6%) tumor tissues samples of lung adenocarcinoma patients. Notably, the lung adenocarcinoma patients with a high TSHZ2 expression tended to have EGFR mutations less frequently and a preferable prognosis to those with a lower expression. Conclusion: A high TSHZ2 expression inhabited cell proliferation and predicted a better prognosis of lung adenocarcinoma, possibly representing a useful therapeutic target for lung adenocarcinoma.

Phox2b Immunohistochemical Staining in Detecting Enteric Neural Crest Cells in Hirschsprung Disease.

BACKGROUND: It can be challenging to recognize undifferentiated/immature ganglion cells, especially single forms. Ganglion cells and glia are derived from enteric neural crest cells (ENCCs), a group of autonomic nervous system (ANS)-lineage neural crest progenitors that PHOX2B regulates. Phox2b is an excellent marker for neoplastic and non-neoplastic ANS cells (eg, peripheral neuroblastic tumors [pNTs]). We hypothesized that Phox2b immunohistochemical staining (IHC) would also be useful for detecting ENCCs. METHODS: Hematoxylin and eosin, calretinin IHC, and Phox2b IHC were reviewed on 21 pull-through specimens and on a cohort of 12 rectal biopsies. RESULTS: Phox2b IHC demonstrated nuclear positivity in all of the ganglion cells across the different phases of differentiation without background staining. The Phox2b result correlated with the morphological findings, calretinin IHC results, and diagnoses based on the routine diagnostic method. The intensity was uniformly strong in the undifferentiated/immature forms and became variable in the mature forms; this pattern was similar to that seen in pNTs. CONCLUSION: Phox2b IHC was highly sensitive and specific for detecting ganglion cells. It worked especially well for immature ganglion cells, seen in premature neonates, and scattered single forms in transition zones. In basic research settings, Phox2b can be a useful marker for early differentiation of ENCCs.

Breast Cancer Index Predicts Extended Endocrine Benefit to Individualize Selection of Patients with HR+ Early-stage Breast Cancer for 10 Years of Endocrine Therapy.

PURPOSE: Individualized selection of patients with early-stage hormone receptor-positive (HR+) breast cancer for extended endocrine therapy (EET) is required to balance modest gains in outcome with toxicities of prolonged use. This study examined the Breast Cancer Index [BCI; HOXB13/IL17BR ratio (H/I)] as a predictive biomarker of EET benefit in patients from the Investigation on the Duration of Extended Adjuvant Letrozole trial. EXPERIMENTAL DESIGN: BCI was tested in primary tumor specimens from 908 patients randomized to receive 2.5 versus 5 years of extended letrozole. The primary endpoint was recurrence-free interval. Cox models and likelihood ratios tested the interaction between EET and BCI (H/I). RESULTS: BCI (H/I)-high significantly predicted benefit from extended letrozole in the overall cohort [HR 0.42; 95% confidence interval (CI), 0.21-0.84; P = 0.011] and any aromatase inhibitor subset [HR 0.34; 95% CI, 0.16-0.73; P = 0.004), whereas BCI (H/I)-low patients did not derive significant benefit (HR 0.95; 95% CI, 0.58-1.56; P = 0.84 and HR 0.90; 95% CI, 0.53-1.55; P = 0.71, respectively) treatment to biomarker interaction was significant (P = 0.045, P = 0.025, respectively). BCI identified approximately 50% of patients with clinically high-risk disease that did not benefit, and with clinically low-risk disease that derived significant benefit, from an additional 2.5 years of EET. CONCLUSIONS: BCI (H/I) predicted preferential benefit from 5 versus 2.5 years of EET and identified patients with improved outcomes from completing 10 years of adjuvant endocrine therapy. Findings expand the clinical utility of BCI (H/I) to a broader range of patients and beyond prognostic risk factors as a predictive endocrine response biomarker for early-stage HR+ breast cancer.

NKX3.1 expression in cervical 'adenoid basal cell carcinoma': another gynaecological lesion with prostatic differentiation?

Adenoid basal cell carcinoma (ABC) is considered a rare cervical neoplasm which when present in 'pure' form, uniquely amongst apparently malignant cervical tumours, has never been reported to metastasise or lead to fatal patient outcome. We recently encountered a case of ABC that was morphologically reminiscent of prostatic differentiation, more specifically basal cell hyperplasia of the prostate. Immunohistochemistry was strongly positive for the prostate related marker NKX3.1 in the glandular cells, but there was no expression of prostate specific antigen (PSA) or prostatic acid phosphatase (PAP). However, subsequent review of five additional cervical ABCs demonstrated focal PAP expression in two of four tested cases, and all were NKX3.1 positive. NKX3.1 expression was also demonstrated in the glandular epithelium of 10 additional gynaecological lesions considered to show prostatic differentiation including five cases of cervical ectopic prostatic tissue, three ovarian teratomas with prostatic differentiation, and two vaginal tubulosquamous polyps. We suggest that some lesions traditionally classified as ABC may in fact represent a variant of prostatic differentiation within the cervix, possibly analogous to basal cell hyperplasia of the prostate.

Distantly Metastatic Retinoblastoma to Soft Tissue and Bone: A Challenging Diagnosis Highlighting the Utility of CRX.

Distant metastasis of retinoblastoma to sites outside the central nervous system is rare; such cases may present years following primary treatment. Diagnosis may be difficult given the rarity of such events and considerable histologic mimics. We describe the clinicopathologic features of 6 cases of metastatic retinoblastoma to distant bone and soft tissue sites from 2 large academic centers. Patients were 3 female and 3 male children; median age was 9.5 years (range: 5 to 15 y) with a mean interval from primary disease diagnosis of 8.0 years (range: 0.75 to 14 y). Metastasis to bones of the lower extremities was most common, occurring in 4 of 6 cases. Tumors showed typical histologic features of retinoblastoma, with sheets of primitive round cells with minimal cytoplasm and indistinct nucleoli; however, characteristic Flexner-Wintersteiner rosettes were absent. A subset of cases demonstrated an alveolar growth pattern, and 2 cases showed higher grade cytology with nuclear anaplasia and prominent nucleoli. Immunohistochemistry for CRX and RB1 showed uniform positivity and loss of expression, respectively. Metastatic retinoblastoma outside the central nervous system may present following long disease-free intervals. Immunohistochemistry for CRX is helpful to confirm this challenging diagnosis.

Utility of NKX3.1 immunohistochemistry in the differential diagnosis of seminal vesicles versus prostatic tissue in needle biopsy.

NKX3.1 is considered a reliable immunohistochemical marker of prostatic origin with high specificity and sensitivity. However, NKX3.1 positivity has been described in other neoplastic and non-neoplastic tissues, such as mesenchymal chondrosarcoma, sex-cord stromal tumors, rete testis adenocarcinoma, lobular and ductal carcinoma of the breast, salivary glands, peribronchial submucosal glands, and Sertoli cells. We analyzed expression of two antibodies (mono and polyclonal) of NKX3.1 in a total of 63 non-neoplastic seminal vesicles. We used 52 resection materials (12 seminal vesicles without prostatic adenocarcinoma, 26 seminal vesicles with prostatic adenocarcinoma infiltration, and 14 cases of seminal vesicles infiltrated by urothelial carcinoma) and 11 prostatic core needle biopsies with incidentally sampled fragment of seminal vesicles. In all cases, tissues from seminal vesicles were completely negative for NKX3.1, despite using polyclonal and monoclonal NKX3.1 antibodies, and regardless of the detection system utilized (diaminobenzidine (DAB) versus alkaline phosphatase (AF)). However, prostatic adenocarcinoma was negative in several cases (n = 6), when AF detection system was used. Reaction with DAB was strong and robust in all cases. Based on our data, we can recommend NKX3.1 as a negative immunohistochemical marker of seminal vesicles.

Clinicopathologic Study of Gleason Pattern 5 Prostatic Adenocarcinoma With "Single-cell" Growth Reveals 2 Distinct Types, One With "Plasmacytoid" Features.

Each Gleason score category of prostatic adenocarcinoma (or Grade Group) may encompass a diverse group of architectural patterns such as well-formed glands, poorly formed glands, cribriform structures, single cells, and/or solid sheets. We have noted heterogeneity within the single-cell subtype of Gleason pattern 5 prostatic adenocarcinoma that has not been fully addressed. Therefore, we retrospectively reviewed a series of radical prostatectomies with high-grade prostatic adenocarcinoma (Grade Group 4 or 5), identifying tumors with a component of single-cell infiltration. Additional cases identified prospectively were also included. TNM status, association with other histologic patterns, and clinical follow-up status were determined. Immunohistochemistry for NKX3.1, E-cadherin, p120 catenin, and prostate-specific antigen (PSA) were performed in each case. Eighteen cases with a component of well-developed Gleason pattern 5 characterized by single infiltrative cells that comprised ≥5% of the tumor were identified (15/202 retrospective radical prostatectomies with the high-grade disease [7.5%]). The single-cell pattern ranged from 5% to 50% of the tumor volume, with 5 cases containing ≥40%, and variable secondary architecture included diffuse infiltrating single cells with targetoid growth pattern around benign glands, solid expansive nests of noncohesive cells, and corded/single file growth pattern. Further morphologic analysis demonstrated 2 distinct histologic subtypes: (1) (subtype 1; n=9) monomorphic "plasmacytoid" tumor cells with eccentrically placed nuclei and variable intracytoplasmic vacuoles with bland cytology and discohesion and (2) (subtype 2; n=9) more cohesive tumor cells with greater cytologic atypia characterized by prominent nucleoli, greater variability in nuclear size/shape, occasional mitotic figures, and more irregular infiltration. By immunohistochemistry, NKX3.1 nuclear expression and PSA cytoplasmic expression was retained in all cases. Concomitant membranous E-cadherin loss and strong cytoplasmic p120 catenin expression were present in 5 of the 18 (28%) cases, all in subtype 1 (5/9, 56%). Overall, 56% (10/18) of patients had advanced-stage disease (≥pT3b), and 70% (7/10) of these patients had associated lymphovascular invasion. All patients had concomitant cribriform patterns of carcinoma. The outcome was available for 14 patients: 4 died of unknown cause; 6 had biochemical recurrence with distant bone metastasis in 5 of the 6; and 4 patients with <3 years of follow-up currently have undetectable serum PSA levels (2 patients received salvage radiotherapy with androgen deprivation and 2 remain on routine follow-up). In summary, the single-cell pattern of Gleason pattern 5 prostatic adenocarcinoma is uniformly associated with other high-risk histologic patterns (eg, cribriform growth), and high-stage disease with distant metastasis is not uncommon. Our data suggest that the "single-cell" Gleason pattern 5 prostatic adenocarcinoma contains 2 distinct subtypes. Somatic CDH1 alterations may play a role in the development of the "plasmacytoid" pattern characterized by monomorphic cytology with concomitant E-cadherin loss and aberrant p120 catenin expression.

PHOX2B Immunostaining: A Simple and Helpful Tool for the Recognition of Ganglionic Cells and Diagnosis of Hirschsprung Disease.

Hirschsprung disease (HD) is a congenital disorder of the enteric nervous system that occurs in ∼1 in 5000 live births. It is characterized by the absence of ganglionic cells (GCs) in the distal intestine. The diagnosis relies on the thorough analysis of a rectal suction biopsy (RSB), which must show a complete absence of GCs after careful examination of at least 100 serial sections. Such a negative characteristic explains the difficulty of this diagnosis. Moreover, GCs may be immature in very young or preterm born children, making them hard to recognize. Therefore, ancillary techniques have been developed as diagnostic help, such as acetylcholinesterase histochemistry and calretinin immunostaining. These techniques reveal only indirect clues, focusing mainly on the changes in nerve fibers, but not on GCs themselves. As PHOX2B has been shown to be a very specific transcription factor in GCs and in progenitor enteric nerve cells, we have assessed (i) PHOX2B immunostaining in immature enteric ganglia and (ii) the use of PHOX2B immunostaining for the recognition of GCs on RSBs for suspicion of HD. We have observed PHOX2B expression in all GCs, both mature and immature, and its complete absence in Hirschsprung cases. We suggest that the use of PHOX2B immunostaining is of great help (i) in the recognition of GCs on RSBs regardless of their differentiation and therefore (ii) in the diagnosis of HD.

Engrailed Homeobox 1 and Cytokeratin 19 Are Independent Diagnostic Markers of Eccrine Porocarcinoma and Distinguish It From Squamous Cell Carcinoma.

OBJECTIVES: The diagnostic utility of En1 in the histopathologic differentiation of eccrine porocarcinoma (EPC) from invasive squamous cell carcinoma (SCC) was investigated. METHODS: Expression of En1 and CK19 in 16 cases of EPC was immunohistochemically examined and compared with that in 32 cases of SCC. RESULTS: In all 16 EPCs, En1 was expressed in 3% to 100% of tumor cells. In 20 of the 32 SCCs, En1 was expressed in 3% to 90% of tumor cells. A total of 13 of the 16 EPCs and five of the 32 SCCs were judged as En1 positive, with a cutoff value of 25%. In addition, 11 of the 16 EPCs and four of the 32 SCCs were CK19 positive. The frequencies of En1- and CK19-positive cases were significantly higher in EPCs than in SCCs. In a logistic regression analysis for predicting EPC, En1 and CK19 were independent markers. When expression patterns of En1 and CK19 were combined, none of the 32 SCCs was both positive. In contrast, 15 of the 16 EPCs were positive for either En1 or CK19. CONCLUSIONS: A combination of En1 and CK19 expression can improve the accuracy of histologic diagnosis of EPC.