Hormone receptor expression in hypospadias.

INTRODUCTION: Sex hormone imbalance in utero is hypothesized to play an important role in the pathogenesis of hypospadias. Due to its easy accessibility, foreskin samples have been used to describe hormone receptor expression in rodents, and both adult and pediatric patients. In this study we conducted a systematic approach to assess hormone receptor expression in pediatric patients with hypospadias compared to healthy controls with a focus on age-matching and differences in severity and degree of hypospadias. METHODS: Foreskin samples were collected from 35 children during hypospadias operations (18 distal and 17 proximal hypospadias) and compared with ventral foreskin samples of a control group of 32 children during circumcision (15 age-matched and 17 older boys). The samples were stained with H/E, androgen (AR), estrogen (ER) and progesterone receptors (PR). The receptor stainings were blindly evaluated. An Allred score was used to evaluate receptor expression in both the epithelium as well as stroma. RESULTS: AR was detected in all cases. AR expression in the stroma was more evident than in the epithelium. AR expression in the hypospadias groups was significantly less than the age matched controls (p < 0.05). There was no significant difference between the two hypospadias groups nor between the two control groups. Older control group showed significantly elevated levels of AR expression compared to the hypospadias group (p < 0.05). ER was also detected in all cases. The stroma showed more ER than in epithelium. PR was minimal or negative in all samples. CONCLUSION: Boys with hypospadias showed significantly weaker expression of androgen receptors than age matched controls. The severity of hypospadias did not influence hormone receptor distribution. AR expression is better observed in the stroma than in the epithelium. There was no difference in ER expression between the hypospadias group (distal or proximal) and age matched normal controls. ER was expressed in larger numbers in normal older preputial tissue. The foreskin of prepubertal boys shows little to no expression of PR.

Comparison between cone-beam breast-CT and full-field digital mammography for microcalcification detection depending on breast density.

The purpose of this study was to evaluate the impact of breast density on the diagnostic performance of cone-beam breast-CT (CBBCT) in comparison to full-field digital mammography (FFDM) for the detection of microcalcifications. This retrospective IRB-approved study was conducted between December 2015 and March 2017 and enrolled 171 women with Breast Imaging Reporting and Data System category 4 or 5 lesions on FFDM and additional CBBCT; 56 of which were ineligible. The inclusion was restricted to 83 women (90 breasts, 90 lesions) with microcalcifications. All lesions underwent histology or were monitored by FFDM and a clinical examination at least 2 years after enrollment. Two breast radiologists independently read each data set twice. Sensitivity, specificity and area under the curve were compared between the modalities. Thirty-two breasts (35.5%) were grouped as non-dense breasts (American College of Radiology types a/b) and 58 breasts (64.5%) as dense breasts (American College of Radiology types c/d). Histopathological assessment was performed in 61 of 90 breast lesions (32 malignant, 1 high-risk and 28 benign). Area under the curve was larger for FFDM than for CBBCT (P = .085). The sensitivity was significantly higher for FFDM compared to CBBCT (P = .009). The specificity showed no significant differences comparing FFDM (both readers: 0.62) versus CBBCT (reader 1: 0.76, reader 2: 0.60; P = .192). Inter-observer-reliability on BI-RADS readings was almost perfect for FFDM and moderate for CBBCT (κ = 0.84, κ = 0.54, respectively). Intra-observer agreement was substantial to almost perfect for both methods and readers. Compared with FFDM, CBBCT demonstrated non-comparable results for microcalcification detection in dense and non-dense breasts.

The urethral plate and the underlying tissue; a histological and histochemical study.

OBJECTIVE: To examine the urethral plate and the underlying tissues in children with proximal hypospadias associated with severe chordee. MATERIALS AND METHODS: The urethral plate and the underlying tissue specimens were excised to correct severe chordee in 17 children with proximal and perineal hypospadias with severe chordee. The median age was 20 months (range 8-36). Sections samples were marked and examined from proximal to distal. Specimens were examined histologically using hematoxylin-eosin (H/E) and Elastic van Gieson (EvG) stain. Histochemical examination was also performed using smooth muscle actin (SMA) and factor 8 antibodies. For control, samples from four patients with hypoplastic urethra proximal to the meatus including the hypoplastic segments until the normal urethra were taken. In addition, the urethra of an adult patient with penile tumor was used as control. RESULTS: The average size of the 17 tissue samples was 0.5 cm × 0.5 cm x 0.3 cm in depth. There was a common pattern that was seen in all the 17 specimens with a variable degree of expression. H/E staining showed that the epithelial lining changed from pseudostratified epithelium at the proximal intact urethra to non-keratinized stratified squamous epithelium at the urethral meatus to keratinized stratified squamous epithelium distally at the urethral plate level. EvG staining showed overall very few elastic fibres that increased slightly in the distal urethral plate. SMA staining showed a circular pattern of smooth muscle cells in the proximal intact urethra that changed to a U-shaped pattern at the level of the meatus, to a triangle shaped pattern just distal to the meatus. The distal urethral plate showed an irregular, disorganized rather flat pattern of the smooth muscles. Factor 8 antibodies staining the blood spaces revealed dysplastic unorganized large blood sinusoids underneath the urethral plate that were different from normal capillaries surrounding the proximal urethra. CONCLUSION: The urethral plate and the underlying tissues in patients with severe chordee have different structure from normal urethra as compared to available literature and the adult control patient. The lack of elastic fibres may help to explain the rigidity of the ventral penis causing chordee. The disorganized irregular distribution of the smooth muscle fibres is suggestive of the hypoplastic corpus spongiosum. The abnormal large blood sinusoids may explain the poor healing quality of the ventral penis in patients with perineal and proximal patients associated with severe chordee. This may explain persistent/recurrent chordee observed later in those patients with severe chordee when dorsal plication is used. The study also supports the recent trend of 2 stage procedure as a plan of management for patients with proximal and perineal hypospadias with severe chordee and excision of all the dysplastic tissues during the first operation.

Mucoricin is a ricin-like toxin that is critical for the pathogenesis of mucormycosis.

Fungi of the order Mucorales cause mucormycosis, a lethal infection with an incompletely understood pathogenesis. We demonstrate that Mucorales fungi produce a toxin, which plays a central role in virulence. Polyclonal antibodies against this toxin inhibit its ability to damage human cells in vitro and prevent hypovolemic shock, organ necrosis and death in mice with mucormycosis. Inhibition of the toxin in Rhizopus delemar through RNA interference compromises the ability of the fungus to damage host cells and attenuates virulence in mice. This 17 kDa toxin has structural and functional features of the plant toxin ricin, including the ability to inhibit protein synthesis through its N-glycosylase activity, the existence of a motif that mediates vascular leak and a lectin sequence. Antibodies against the toxin inhibit R. delemar- or toxin-mediated vascular permeability in vitro and cross react with ricin. A monoclonal anti-ricin B chain antibody binds to the toxin and also inhibits its ability to cause vascular permeability. Therefore, we propose the name 'mucoricin' for this toxin. Not only is mucoricin important in the pathogenesis of mucormycosis but our data suggest that a ricin-like toxin is produced by organisms beyond the plant and bacterial kingdoms. Importantly, mucoricin should be a promising therapeutic target.

Of Cestodes and Men: Surgical Treatment of a Spinal Hydatid Cyst.

BACKGROUND: The cestode Echinococcus granulosus causes hydatid disease. In addition to manifestations in the liver and lung, it can lead to cystic lesions in the spine. CASE DESCRIPTION: We report a 42-year-old male patient with primary hydatid disease in the eighth thoracic vertebra. The only clinical symptom was chronic back pain. Although laboratory findings were normal, imaging displayed lytic destruction that raised the suspicion of a metastatic disease. Diagnostics of the thoraces and abdomen did not reveal other pathologic abnormalities. Follow-up magnetic resonance imaging (MRI) depicted a progressive compression of the spinal cord and inhomogeneous structure in the fat-suppressed sequences. Because the Jamshidi biopsy was inconclusive, the tumor board recommended surgery. Dorsal decompression, spondylodesis of T6-T10, and vertebral column resection of T8 with complete cyst removal were performed. The resected vertebrae showed a mucous-like lesion with white granular tissue interfusing the whole vertebral body. A pathologic examination and enzyme-linked immunosorbent assay confirmed E. granulosus. Thus chemotherapy with albendazole was initiated. A follow-up MRI of the whole spine confirmed complete remission and found no additional resettlements. The patient's back pain was resolved without neurologic deficits. CONCLUSIONS: For lytic manifestations of the vertebral column, hydatid cysts should be considered a differential diagnosis in addition to malignant metastasis, tuberculosis, and osteomyelitis. Thorough surgical resection and strict follow-up are necessary.

Breast lesion size assessment in mastectomy specimens: Correlation of cone-beam breast-CT, digital breast tomosynthesis and full-field digital mammography with histopathology.

To compare the accuracy of breast lesion size measurement of cone-beam breast-CT (CBBCT), digital breast tomosynthesis (DBT) and full-field digital mammography (FFDM).Patients scheduled for mastectomy due to at least 1 malignant breast lesion were included. Mastectomy specimens were examined by CBBCT, DBT, FFDM, and histopathology.A total of 94 lesions (40 patients) were included. Histopathological analyses revealed 47 malignant, 6 high-risk, and 41 benign lesions. Mean histopathological lesion size was 20.8 mm (range 2-100). Mean absolute size deviation from histopathology was largest for FFDM (5.3 ±â€Š6.7 mm) and smallest for CBBCT 50 mA, high-resolution mode (4.3 ±â€Š6.7 mm). Differences between imaging modalities did not reach statistical significance (P = .85).All imaging methods tend to overestimate breast lesion size compared to histopathological gold standard. No significant differences were found regarding size measurements, although in tendency CBBCT showed better lesion detection and cT classification over FFDM.

Generation of highly differentiated BHY oral squamous cell carcinoma multicellular spheroids.

Three-dimensional (3D) multicellular spheroids (MCS) are considered suitable models in cancer research and anticancer drug development. Although studying the complex tumour characteristics from all different degrees of malignancy is vital, MCS generation has only been described in a few moderately- and poorly differentiated oral squamous cell carcinoma (OSCC) cell lines. No previous study has demonstrated the MCS formation in a highly differentiated OSCC cell line. For the first time, the present study aimed to generate MCS from the highly differentiated OSCC cell line BHY. BHY spheroids were grown in three independent experiments in 96-well plates through the use of the liquid overlay technique. Although BHY cells are grow slowly and are difficult to culture, they formed compact MCS within 24 h. After 3 days of incubation, no further increase in spheroid size was observed. MCS were harvested, paraffin-embedded and 2 µm tissue sections were prepared for further analysis. The diameter and volume of each spheroid were determined. BHY MCS diameter ranged between 46.76 and 233.26 µm, with a volume range from 5.35×104-6.65×106 µm3. In conclusion, using the liquid overlay technique, the highly differentiated OSCC cell line BHY forms different sized spheroids, which may be used for further investigations.

High mast cell density indicates a longer overall survival in oral squamous cell carcinoma.

This study evaluates the effects of tumour-associated mast cells on the prognosis of patients suffering from oral squamous cell carcinoma (OSCC). Tryptase-positive (MCT+) and CD117-positive (CD117+) mast cells were immunohistochemically evaluated in tissue samples of 118 OSCC patients. Besides, various clinicopathological parameters, the influence of the MCT+ and CD117+ mast cell density on overall survival and the incidence of first local recurrence was analysed by Cox regression and competing risk regression. Among all investigated parameters, multiple Cox regression revealed a significant influence of the MCT+ (cut-off at 14.87 mast cells/mm2 stroma; p = 0.0027) and CD117+ mast cell density (cut-off at 33.19 mast cells/mm2 stroma; p = 0.004), the age at primary diagnosis, and the T and N stage (all p-values < 0.05) on overall survival. Patients with a low mast cell density showed a significantly poorer overall survival rate compared to those with a high mast cell density in the tumour-associated stroma. Competing risk regression revealed a significant influence of the resection status (R) on the incidence of first local recurrence (p = 0.0023). A high mast cell density in the tumour-associated stroma of oral squamous cell carcinoma indicates a longer patient survival.

Contrast Enhancement on Cone-Beam Breast-CT for Discrimination of Breast Cancer Immunohistochemical Subtypes.

PURPOSE: To evaluate whether contrast enhancement on cone-beam breast-CT (CBBCT) could aid in discrimination of breast cancer subtypes and receptor status. METHODS: This study included female patients age >40 years with malignant breast lesions identified on contrast-enhanced CBBCT. Contrast enhancement of malignant breast lesions was standardized to breast fat tissue contrast enhancement. All breast lesions were approved via image-guided biopsy or surgery. Immunohistochemical staining was conducted for expression of estrogen (ER), progesterone (PR), human epidermal growth factor receptor-2 (HER2) and Ki-67 index. Contrast enhancement of breast lesions was correlated with immunohistochemical breast cancer subtypes (Luminal A, Luminal B, HER2 positive, triple negative), receptor status and Ki-67 expression. RESULTS: Highest contrast enhancement was seen for Luminal A lesions (93.6 HU) compared to Luminal B lesions (47.6 HU, P=.002), HER2 positive lesions (83.5 HU, P=.359) and triple negative lesions (45.3 HU, P=.005). Contrast enhancement of HER2 positive lesions was higher than Luminal B lesions (P=.044) and triple negative lesions (P=.039). No significant difference was evident between Luminal B and triple negative lesions (P=.439). Lesions with high Ki-67 index showed lower contrast enhancement than those with low Ki-67 index (P=.0043). ER, PR and HER2 positive lesions demonstrated higher contrast enhancement than their receptor negative counterparts, although differences did not reach statistical significance (P=.1714; P=.3603; P=.2166). CONCLUSIONS: Contrast enhancement of malignant breast lesions on CBBCT correlates with immunohistochemical subtype and proliferative potential. Thereby, CBBCT might aid in selecting individualized treatment strategies for breast cancer patients based on pre-operative imaging.

Cone-beam Breast Computed Tomography: CT Density Does Not Reflect Proliferation Potential and Receptor Expression of Breast Carcinoma.

PURPOSE: Recently, cone-beam breast computed tomography (CBCT) is established for the breast investigation. The purpose of the present study was to investigate possible associations between CBCT findings and histopathological features in breast cancer. METHODS: Overall, 59 female patients, mean age of 64.6 years with histological proven breast cancer were included into the study. In all cases, non-contrast CBCT examination was done. The diagnosis of the identified lesions was confirmed histologically by biopsy. Immunohistochemical staining against estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki-67 was performed for every lesion. Collected data were evaluated by means of descriptive statistics. Spearman's correlation coefficient was used to analyze the association between CT density and Ki-67 values. P values <0.05 were taken to indicate statistical significance in all instances. RESULTS: The size of the lesion varied from 2.7 to 90.0, mean size, 15.88±13.0 mm. The mean value of CT density of the lesions was 63.95±38.18 HU. The density tended to be higher in tubular carcinoma. Correlation analysis identified no significant correlations between CT density and Ki-67 level (r=-0.031, P=.784). There were no statistically significant differences of CT density between tumors with different receptor status. CONCLUSIONS: No significant associations between CT density and receptor status in breast cancer. Tubular carcinoma tended to have higher CT density in comparison to other subtypes of breast carcinomas.

The Early Adaptive Immune Response in the Pathophysiological Process of Pneumococcal Meningitis.

BACKGROUND: The adaptive immune system has been considered to play a minimal role in the early host response during bacterial meningitis. METHODS: We investigated the progression and outcome of pneumococcal meningitis in Rag1-/- mice lacking functional B and T cells by assessing overall and symptom-free survival, bacteriological and histological studies, as well as flow cytometry and measurements of proinflammatory mediators. RESULTS: The intracerebral injection of S. pneumoniae D39 induced the recruitment of B and T cells (CD4+, γδ and natural killer) into the brain of wild-type mice. Mice with no functional B and T cells developed clinical symptoms and succumbed to the infection earlier than the wild-type group. In the CNS, Rag1-/- mice showed lower levels of interleukin 1ß, reduced microglial proliferation, and impaired granulocyte recruitment with an earlier spread of pneumococci into the bloodstream, compared with wild-type mice. Lack of B and T cells resulted in a severe impairment of bacterial clearance in blood, spleen, and liver and an exaggerated systemic inflammatory response. CONCLUSIONS: B and T cells are important effector cells delaying the spread of pneumococci from the brain to the systemic circulation and shaping the immune response, thereby prolonging the survival of the host in the absence of antibiotic treatment.

FLT3-ITD and TLR9 use Bruton tyrosine kinase to activate distinct transcriptional programs mediating AML cell survival and proliferation.

Acute myeloid leukemia (AML) is driven by niche-derived and cell-autonomous stimuli. Although many cell-autonomous disease drivers are known, niche-dependent signaling in the context of the genetic disease heterogeneity has been difficult to investigate. Here, we analyzed the role of Bruton tyrosine kinase (BTK) in AML. BTK was frequently expressed, and its inhibition strongly impaired the proliferation and survival of AML cells also in the presence of bone marrow stroma. By interactome analysis, (phospho)proteomics, and transcriptome sequencing, we characterized BTK signaling networks. We show that BTK-dependent signaling is highly context dependent. In Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD)-positive AML, BTK mediates FLT3-ITD-dependent Myc and STAT5 activation, and combined targeting of FLT3-ITD and BTK showed additive effects. In Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD)-negative AML, BTK couples Toll-like receptor 9 (TLR9) activation to nuclear factor κΒ and STAT5. Both BTK-dependent transcriptional programs were relevant for cell cycle progression and apoptosis regulation. Thus, we identify context-dependent oncogenic driver events that may guide subtype-specific treatment strategies and, for the first time, point to a role of TLR9 in AML. Clinical evaluation of BTK inhibitors in AML seems warranted.

The BCL9-2 proto-oncogene governs estrogen receptor alpha expression in breast tumorigenesis.

The majority of human breast cancers express estrogen receptor alpha (ER), which is important for therapy with anti-estrogens. Here we describe the role of BCL9-2, a proto-oncogene previously characterized as co-activator of Wnt/ß-catenin signaling, for mammary tumorigenesis in mice and human. ER positive human breast cancers showed overexpression of BCL9-2 and tamoxifen treated patients with high BCL9-2 demonstrated a better survival. BCL9-2 was upregulated during puberty and pregnancy in normal mammary epithelia, but downregulated in the involuted gland. BCL9-2 overexpression in vivo delayed the mammary involution and induced alveolar hyperplasia. Moreover, aged BCL9-2 transgenic mice developed ductal-like mammary tumors with high nuclear ER expression. We found, that primary cell cultures of BCL9-2 breast tumors responded to tamoxifen treatment. Moreover, BCL9-2 regulated the expression of ER and the proliferation of human breast cancer cells independently of ß-catenin. Finally, we describe a novel mechanism, how BCL9-2 regulates ER transcription by interaction with Sp1 through the proximal ESR1 gene promoter. In summary, BCL9-2 induces ER positive breast cancers in vivo, regulates ER expression by a novel ß-catenin independent mechanism in breast cancer cells, and might predict the therapy response to tamoxifen treatment.

Membrane connexin 43 acts as an independent prognostic marker in oral squamous cell carcinoma.

The aim of the present study was to evaluate the expression and localization of connexin (Cx) 26, -43 and -45 in a group of 35 patients with primary oral squamous cell carcinoma (OSCC) with the objective of making a more accurate disease prognosis. We analysed the expression of connexins in tissue samples of primary OSCC, matching oral mucosa free of dysplasia, and its associated lymph node metastases (LNM) by semi-quantitative immunohistochemistry of membrane, cytoplasmic and nuclear connexin expression. The levels of expression were correlated with the overall survival time (OS). Cx43 was overexpressed in tumour cells compared to epithelia in dysplasia-free mucosa. High membrane expression of Cx43 on tumour cells was the only statistically significant and independent prognostic factor of short OS (P=0.0088). Membrane expression of Cx43 in matching dysplasia-free mucosa acted similarly, but did not reach statistical significance (P=0.059). No correlation was found between the Cx26, Cx45 expression and OS. We conclude that Cx43 expression in dysplasia-free mucosa may indicate a very early stage of tumour promotion. Although overexpression of Cx43 is found in invasive tumours we only found membrane Cx43 expression to correlate with OS. This observation suggests that cytoplasmic Cx43 serves as storage and only membrane translocation may promote the formation of gap junctions and gap junctional intercellular communication (GJIC) with prognostic relevance.

Mandibular reconstruction using a calcium phosphate/polyethylene glycol hydrogel carrier with BMP-2.

AIM: To test the hypothesis that a synthetic hydroxyapatite/ß-tricalcium phosphate (HA/TCP) construct combined with polyethylene glycol (PEG) hydrogel including recombinant human bone morphogenetic proteins-2 (rhBMP-2) enhances new bone formation compared with bone morphogenetic proteins-2 (BMP-2) delivered using the HA/TCP construct alone. MATERIAL AND METHODS: Bilateral mandibular partial thickness 20 × 8 × 8 mm (L × W × H) alveolar defects were surgically created in the edentulated posterior mandible in 18 female minipigs. Randomized into two groups of nine animals each, the alveolar defects either received HA/TCP or HA/TCP/PEG with or without BMP-2 (105 µg/defect) in contra-lateral sites using a split-mouth design. Primary outcome, bone density (%) within four regions of interest, was evaluated following a 4-week healing interval when the animals were killed for histometric analysis. RESULTS: Bone morphogenetic proteins-2 loaded onto HA/TCP constructs significantly enhanced new bone formation compared with HA/TCP controls. Adding PEG apparently obstructed BMP-2 induced bone formation. CONCLUSION: Polyethylene glycol compromises the osteogenic effect of BMP-2.