Different populations of A(H1N1)pdm09 viruses in a patient with hemolytic-uremic syndrome.

Respiratory viral infections may have different impacts ranging from infection without symptoms to severe disease or even death though the reasons are not well characterized. A patient (age group 5-15 years) displaying symptoms of hemolytic uremic syndrome died one day after hospitalization. qPCR, next generation sequencing, virus isolation, antigenic characterization, resistance analysis was performed and virus replication kinetics in well-differentiated airway cells were determined. Autopsy revealed hemorrhagic pneumonia as major pathological manifestation. Lung samples harbored a large population of A(H1N1)pdm09 viruses with the polymorphism H456H/Y in PB1 polymerase. The H456H/Y viruses replicated much faster to high viral titers than upper respiratory tract viruses in vitro. H456H/Y-infected air-liquid interface cultures of differentiated airway epithelial cells did reflect a more pronounced loss of ciliated cells. A different pattern of virus quasispecies was found in the upper airway samples where substitution S263S/F (HA1) was observed. The data support the notion that viral quasispecies had evolved locally in the lung to support high replicative fitness. This change may have initiated further pathogenic processes leading to rapid dissemination of inflammatory mediators followed by development of hemorrhagic lung lesions and fatal outcome.

B-cell receptor reactivity against Rothia mucilaginosa in nodular lymphocyte-predominant Hodgkin lymphoma.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a Hodgkin lymphoma expressing functional B-cell receptors (BCR). Recently, we described a dual stimulation model of IgD+ lymphocyte-predominant cells by Moraxella catarrhalis antigen RpoC and its superantigen MID/hag, associated with extralong CDR3 and HLA-DRB1*04 or HLADRB1* 07 haplotype. The aim of the present study was to extend the antigen screening to further bacteria and viruses. The fragment antibody-binding (Fab) regions of seven new and 15 previously reported cases were analyzed. The reactivity of non-Moraxella spp.-reactive Fab regions against lysates of Rothia mucilaginosa was observed in 5/22 (22.7%) cases. Galactofuranosyl transferase (Gltf) and 2,3-butanediol dehydrogenase (Bdh) of R. mucilaginosa were identified by comparative silver- and immuno-staining in two-dimensional gels, with subsequent mass spectrometry and validation by western blots and enzyme-linked immunosorbent assay. Both R. mucilaginosa Gltf and Bdh induced BCR pathway activation and proliferation in vitro. Apoptosis was induced by recombinant Gltf/ETA'-immunotoxin conjugates in DEV cells expressing recombinant R. mucilaginosa-reactive BCR. Reactivity against M. catarrhalis RpoC was confirmed in 3/7 newly expressed BCR (total 10/22 reactive to Moraxella spp.), resulting in 15/22 (68.2%) cases with BCR reactivity against defined bacterial antigens. These findings strengthen the hypothesis of bacterial trigger contributing to subsets of NLPHL.

THSD7A Positivity Predicts Poor Survival and Is Linked to High FAK Expression and FGFR1-Wildtype in Female Patients with Squamous Cell Carcinoma of the Lung.

Lung cancer is the leading cause of cancer-related deaths in the western world, with squamous cell carcinoma being one of the most common histological subtypes. Prognostic and predictive markers are still largely missing for squamous cell carcinoma of the lung (LSCC). Several studies indicate that THSD7A might at least play a role in the prognosis of different tumors. FAK seems to play an important role in lung cancer and is discussed as a potential therapeutic target. In addition, there is evidence that FAK-dependent signaling pathways might be affected by THSD7A. For that reason, we investigated the role of THSD7A as a potential tumor marker in LSCC and whether THSD7A expression has an impact on the expression level of FAK. A total of 101 LSCCs were analyzed by immunohistochemistry using tissue microarrays. THSD7A positivity was associated with poor overall survival in female patients and showed a relation to high FAK expression in this subgroup. To our knowledge, we are the first to report these correlations in lung cancer. The results might be proof of the assumed activation of FAK-dependent signaling pathways by THSD7A and that as a membrane-associated protein, THSD7A might serve as a putative therapeutic target in LSCC.

The 3q Oncogene SEC62 Predicts Response to Neoadjuvant Chemotherapy and Regulates Tumor Cell Migration in Triple Negative Breast Cancer.

In the absence of targeted treatment options, neoadjuvant chemotherapy (NACT) is applied widely for triple-negative breast cancer (TNBC). Response to NACT is an important parameter predictive of oncological outcomes (progression-free and overall survival). An approach to the evaluation of predictive markers enabling therapy individualization is the identification of tumor driver genetic mutations. This study was conducted to investigate the role of SEC62, harbored at 3q26 and identified as a driver of breast cancer pathogenesis, in TNBC. We analyzed SEC62 expression in The Cancer Genome Atlas database, and immunohistologically investigated SEC62 expression in pre- and post-NACT tissue samples from 64 patients with TNBC treated at the Department of Gynecology and Obstetrics/Saarland University Hospital/Homburg between January 2010 and December 2018 and compared the effect of SEC62 on tumor cell migration and proliferation in functional assays. SEC62 expression dynamics correlated positively with the response to NACT (p ≤ 0.01) and oncological outcomes (p ≤ 0.01). SEC62 expression stimulated tumor cell migration (p ≤ 0.01). The study findings indicate that SEC62 is overexpressed in TNBC and serves as a predictive marker for the response to NACT, a prognostic marker for oncological outcomes, and a migration-stimulating oncogene in TNBC.

Immunohistochemical assessment of PD-L1 expression using three different monoclonal antibodies in triple negative breast cancer patients.

BACKGROUND: PD-L1 receptor expression in breast cancer tissue can be assessed with different anti-human PD-L1 monoclonal antibodies. The performance of three specific monoclonal antibodies in a head-to-head comparison is unknown. In addition, a potential correlation of PD-L1 expression and clinico-pathological parameters has not been investigated. METHODS: This was a retrospective study on tissue samples of patients with histologically confirmed triple negative breast cancer (TNBC). PD-L1 receptors were immune histochemically stained with three anti-human PD-L1 monoclonal antibodies: 22C3 and 28-8 for staining of tumor cell membranes (TC) and cytoplasm (Cyt), SP142 for immune cell staining (IC). Three different tissue samples of each patient were evaluated separately by two observers in a blinded fashion. The percentage of PD-L1 positive tumor cells in relation to the total number of tumor cells was determined. For antibodies 22C3 and 28-8 PD-L1 staining of 0 to < 1% of tumor cells was rated "negative", 1-50% was rated "positive" and > 50% was rated "strong positive". Cyt staining was defined as "negative" when no signal was observed and as "positive", when any positive signal was observed. For IC staining with SP142 all samples with PD-L1 expression ≥ 1% were rated as "positive". Finally, the relationship between PD-L1 expression and clinico-pathological parameters was analyzed. RESULTS: Tissue samples from 59 of 60 enrolled patients could be analyzed. Mean age was 55 years. Both the monoclonal antibodies 22C3 and 28-8 had similar properties, and were positive for both TC in 13 patients (22%) and for Cyt staining in 24 patients (40.7%). IC staining with antibody SP142 was positive in 24 patients (40.7%), who were also positive for Cyt staining. The differences between TC and Cyt staining and TC and IC staining were significant (p = 0.001). Cases with positive TC staining showed higher Ki67 expression compared to those with negative staining, 40 vs 30%, respectively (p = 0.05). None of the other clinico-pathological parameters showed any correlation with PDL1 expression. CONCLUSIONS: Antibodies 22C3 and 28-8 can be used interchangeably for PD-L1 determination in tumor cells of TNBC patients. Results for Cyt staining with 22C3 or 28-8 and IC staining with SP142 were identical. In our study PD-L1 expression correlates with Ki67 expression but not with OS or DFS.

Simultaneous Bilateral Primary Occlusion of the Ophthalmic Artery due to Florid Giant Cell Arteritis.

PURPOSE: To report a case of simultaneous bilateral ophthalmic artery occlusion in diagnosed giant cell arteritis (GCA). OBSERVATIONS: A 77-year-old male patient presented to the emergency department with simultaneous vision loss in both eyes for 3 hours. Headache at both temples and jaw claudication had been present for 3 weeks. Laboratory values demonstrated an initially increased C-reactive protein (CRP) of 202.0 mg/L and an erythrocyte sedimentation rate (ESR) of 100 mm within the first 20 minutes. Duplex sonography of the right and left temporal arteries revealed a "halo sign." A case of GCA was suspected, and intravenous high-dose methylprednisolone therapy was immediately administered. The clinical examination revealed a bilateral central retinal artery occlusion and fluorescein angiography showed a hot optic disc in the right eye and patchy choroidal hypoperfusion in both eyes. Biopsy of the left temporal artery was performed, which confirmed a florid temporal arteritis with complete thrombotic occlusion of the vascular lumen. Despite a good response to the administered therapy (CRP 17.0 mg/L 1 week after initiation), the visual prognosis was significantly limited through retinal and optic nerve involvement. By the follow-up examination 8 weeks later, the near visual acuity was 20/400 in the right and left eye at a distance of 16 inches. CONCLUSION AND IMPORTANCE: We hereby present a simultaneous bilateral ophthalmic artery occlusion as a rare complication of GCA. The combination of central retinal artery occlusion, arteritic anterior ischemic optic neuropathy, and choroidal hypoperfusion suggests an acute inflammatory involvement of the ophthalmic artery. In cases of the slightest suspicion of giant cell arteritis, an immediate high-dose steroid therapy initiation is of utmost importance.

Blockade of PD-1 decreases neutrophilic inflammation and lung damage in experimental COPD.

Chronic obstructive lung disease (COPD) and lung cancer are both caused by smoking and often occur as comorbidity. The programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) axis is an important canonic immunoregulatory pathway, and antibodies that specifically block PD-1 or PD-L1 have demonstrated efficacy as therapeutic agents for non-small cell lung cancer. The role of the PD-1/PD-L1 axis in the pathogenesis of COPD is unknown. Here, we analyzed the function of the PD-1/PD-L1 axis in preclinical COPD models and evaluated the concentrations of PD-1 and PD-L1 in human serum and bronchoalveolar lavage (BAL) fluids as biomarkers for COPD. Anti-PD-1 treatment decreased lung damage and neutrophilic inflammation in mice chronically exposed to cigarette smoke (CS) or nontypeable Haemophilus influenzae (NTHi). Ex vivo stimulated macrophages obtained from anti-PD-1-treated mice released reduced amounts of inflammatory cytokines. PD-L1 concentrations correlated positively with PD-1 concentrations in human serum and BAL fluids. Lung sections obtained from patients with COPD stained positive for PD-L1. Our data indicate that the PD-1/PD-L1 axis is involved in developing inflammation and tissue destruction in COPD. Inflammation-induced activation of the PD-1 pathway may contribute to disease progression.

Robotic Salvage Lymph Node Dissection in Recurrent Prostate Cancer: Lessons Learned from 68 Cases and Implications for Future Clinical Management.

PURPOSE: Salvage lymph node dissection is a rescue treatment for patients with nodal recurrence after radical prostatectomy. Very limited data are available on robotic salvage lymph node dissection. Our purpose was to investigate perioperative and oncological outcomes of robotic salvage lymph node dissection in a large monocentric series. MATERIALS AND METHODS: Perioperative data, complications within 30 days after surgery and oncological outcomes as assessed by histology, prostate specific antigen changes, prostate specific antigen nadir after salvage lymph node dissection, and time to further therapy were analyzed. To identify predictive factors for oncological outcome, Kaplan-Meier and Cox-regression analyses were performed. For cases with a mismatch between preoperative positron emission tomography/computed tomography and the number of histologically positive lymph nodes, prostate specific membrane antigen immunohistochemistry was performed on removed lymph nodes. RESULTS: A total of 68 patients underwent robotic salvage lymph node dissection with a median operation time of 126 minutes, a blood loss of 50 ml, and a length of stay of 4 days. No major complications (>Clavien 3) occurred. Median followup was 12.1 months. Median time to further therapy was 12.4 months, 37% of patients experienced complete biochemical response (prostate specific antigen <0.2 ng/ml) and 11% reached an undetectable prostate specific antigen, which was maintained for >1 year in 3 cases. Lower preoperative prostate specific antigen, longer time between radical prostatectomy and salvage lymph node dissection, preoperative prostate specific membrane antigen positron emission tomography/computed tomography and complete biochemical response after salvage lymph node dissection were significant predictors of longer therapy-free survival (all p <0.005). Prostate specific membrane antigen immunohistochemistry revealed that prostate specific membrane antigen positron emission tomography/computed tomography tends to miss small lymph node metastases <5 mm. CONCLUSIONS: Robotic salvage lymph node dissection is a feasible approach with low perioperative morbidity and delays further systemic therapy in most patients. Prostate specific membrane antigen positron emission tomography/computed tomography detection is mostly limited to tumor foci >5 mm.

Identification of the atypically modified autoantigen Ars2 as the target of B-cell receptors from activated B-cell-type diffuse large B-cell lymphoma.

It has been suggested that B-cell receptor (BCRs) stimulation by specific antigens plays a pathogenic role in diffuse large B-cell lymphoma (DLBCL). Here, it was the aim to screen for specific reactivities of DLBCL-BCRs in the spectrum of autoantigens and antigens of infectious origin. Arsenite resistance protein 2 (Ars2) was identified as the BCR target of 3/5 ABC-type DLBCL cell lines and 2/11 primary DLBCL cases. Compared to controls, Ars2 was hypo-phosphorylated exclusively in cases and cell lines with Ars2-specific BCRs. In a validation cohort, hypo-phosphorylated Ars2 was found in 8/31 ABC-type, but only 1/20 germinal center B cell (GBC)-like type DLBCL. Incubation with Ars2 induced BCR-pathway activation and increased proliferation, while an Ars2/ETA-toxin conjugate induced killing of cell lines with Ars2-reactive BCRs. Ars2 appears to play a role in a subgroup of ABC-type DLBCLs. Moreover, transformed DLBCL lines with Ars2-reactive BCRs still show growth advantage after incubation with Ars2. These results provide knowledge about the pathogenic role of a specific antigen stimulating the BCR pathway in DLCBL.

Hyper-N-glycosylated SAMD14 and neurabin-I as driver autoantigens of primary central nervous system lymphoma.

To address the role of chronic antigenic stimulation in primary central nervous system lymphoma (PCNSL), we searched for autoantigens and identified sterile α-motif domain containing protein 14 (SAMD14) and neural tissue-specific F-actin binding protein I (neurabin-I) as autoantigenic targets of the B-cell receptors (BCRs) from 8/12 PCNSLs. In the respective cases, SAMD14 and neurabin-I were atypically hyper-N-glycosylated (SAMD14 at ASN339 and neurabin-I at ASN1277), explaining their autoimmunogenicity. SAMD14 and neurabin-I induced BCR pathway activation and proliferation of aggressive lymphoma cell lines transfected with SAMD14- and neurabin-I-reactive BCRs. Moreover, the BCR binding epitope of neurabin-I conjugated to truncated Pseudomonas exotoxin-killed lymphoma cells expressing the respective BCRs. These results support the role of chronic antigenic stimulation by posttranslationally modified central nervous system (CNS) driver autoantigens in the pathogenesis of PCNSL, serve as an explanation for their CNS tropism, and provide the basis for a novel specific treatment approach.

Identification of C/EBPα as a novel target of the HPV8 E6 protein regulating miR-203 in human keratinocytes.

Patients suffering from Epidermodysplasia verruciformis (EV), a rare inherited skin disease, display a particular susceptibility to persistent infection with cutaneous genus beta-human papillomavirus (beta-HPV), such as HPV type 8. They have a high risk to develop non-melanoma skin cancer at sun-exposed sites. In various models evidence is emerging that cutaneous HPV E6 proteins disturb epidermal homeostasis and support carcinogenesis, however, the underlying mechanisms are not fully understood as yet. In this study we demonstrate that microRNA-203 (miR-203), a key regulator of epidermal proliferation and differentiation, is strongly down-regulated in HPV8-positive EV-lesions. We provide evidence that CCAAT/enhancer-binding protein α (C/EBPα), a differentiation-regulating transcription factor and suppressor of UV-induced skin carcinogenesis, directly binds the miR-203 gene within its hairpin region and thereby induces miR-203 transcription. Our data further demonstrate that the HPV8 E6 protein significantly suppresses this novel C/EBPα/mir-203-pathway. As a consequence, the miR-203 target ΔNp63α, a proliferation-inducing transcription factor, is up-regulated, while the differentiation factor involucrin is suppressed. HPV8 E6 specifically down-regulates C/EBPα but not C/EBPß expression at the transcriptional level. As shown in knock-down experiments, C/EBPα is regulated by the acetyltransferase p300, a well-described target of cutaneous E6 proteins. Notably, p300 bound significantly less to the C/EBPα regulatory region in HPV8 E6 expressing keratinocytes than in control cells as demonstrated by chromatin immunoprecipitation. In situ analysis confirmed congruent suprabasal expression patterns of C/EBPα and miR-203 in non-lesional skin of EV-patients. In HPV8-positive EV-lesions both factors are potently down-regulated in vivo further supporting our in vitro data. In conclusion our study has unraveled a novel p300/C/EBPα/mir-203-dependent mechanism, by which the cutaneous HPV8 E6 protein may expand p63-positive cells in the epidermis of EV-patients and disturbs fundamental keratinocyte functions. This may drive HPV-mediated pathogenesis and may potentially also pave the way for skin carcinogenesis in EV-patients.

4-miRNA Score Predicts the Individual Metastatic Risk of Renal Cell Carcinoma Patients.

BACKGROUND: In order to improve individual prognostication as well as stratification for adjuvant therapy in patients with clinically localized clear cell renal cell carcinoma (ccRCC), reliable prognostic biomarkers are urgently needed. In this study, microRNAs (miRNAs) have emerged as promising candidates. We investigated whether a combination of differently expressed miRNAs in primary tumors can predict the individual metastatic risk. METHODS: Using two prospectively collected biobanks of academic centers, 108 ccRCCs were selected, including 57 from patients with metastatic disease at diagnosis or during follow-up and 51 without evidence of metastases. Fourteen previously identified candidate miRNAs were tested in 20 representative formalin-fixed and paraffin embedded samples in order to select the best discriminators between metastatic and nonmetastatic ccRCC. These miRNAs were approved in 108 tumor samples. We evaluated the association of altered miRNA expression with the metastatic potential of tumors using quantitative polymerase chain reaction. A prognostic 4-miRNA model has been established using a random forest classifier. Cox regression analyses were performed for correlation of the miRNA model and clinicopathological parameters to metastasis-free and overall survival. RESULTS: Nine miRNAs indicated significant expression alterations in the small cohort. These miRNAs were validated in the whole cohort. The established 4-miRNA score (miR-30a-3p/-30c-5p/-139-5p/-144-5p) has been identified as a superior predictor for metastasis-free survival (hazard ratio 12.402; p = 7.0E-05) and overall survival (p = 1.1E-04) compared with clinicopathological parameters, and likewise in the Leibovich score subgroups. CONCLUSIONS: We identified a 4-miRNA model that was found to be superior to clinicopathological parameters in accurately predicting individual metastatic risk and can support patient selection for risk-stratified follow-up and adjuvant therapy studies.

Human Papillomavirus-Associated Invasive Condylomas in a Man with Immunosuppressive Comorbidities.

Human papilloma virus (HPV) infections are one of the most common sexually transmitted diseases. We present the case of a 77-year-old Caucasian male with enormous genital warts of the penis, scrotum, groins and anus. Lesions were excised by electrosurgery. The histological examination revealed Condylomata gigantea as well as an invasive perianal squamous cell carcinoma. Mucosal "low-risk" HPV type 6 was detected. The patient had a history of an immunosuppressing disease. During the 4-year follow-up, multiple relapses occurred. Thus, particularly in immunosuppressed patients, early prophylactic HPV vaccination seems to be indicated for use in the prevention of HPV-associated mutilating and life-threatening disease. Vaccination should also protect from "low-risk" HPV.

Sec62/Ki67 dual staining in cervical cytology specimens: a new marker for high-grade dysplasia.

PURPOSE: In the previous studies, we demonstrated that Sec62 is essential for tumor cell migration, epithelial-to-mesenchymal transition, and intracellular stress tolerance. An increase in Sec62 expression correlated with an increase in cervical dysplasia severity in liquid-based cytology specimens. Ki67 is an established proliferation marker. Thus, in this study, we examined a method of Sec62/Ki67 dual staining for the detection of high-grade dysplasia and cancer in cervical liquid-based cytology specimens. METHODS: Sec62/Ki67 dual staining was performed on 100 cervical liquid-based cytology specimens. The staining results were correlated with cytological, immunocytological (p16/Ki67), colposcopic, and histological findings. RESULTS: All 56 (n = 56, 100%) cases of cervical intraepithelial neoplasia grade 3 and cervical cancer (CIN3+ lesions) were positive for Sec62/Ki67 staining, while low-grade lesions and normal cells were negative. Sec62/Ki67 staining was highly sensitive and specific for the detection of CIN2+ and CIN3+ lesions (94.37%; 100% and 100%; 84.09%, respectively). CONCLUSIONS: Sec62/Ki67 dual-staining immunocytochemistry is a promising cytological tool for interpreting high-grade squamous lesions in cytological specimens and for assessing the risk of progression to cancer.

Preoperative morphological diagnosis of axillary lymph nodes in a breast center consultation service: evaluation of fine-needle aspiration and core biopsy techniques.

PURPOSE: Preoperative routine examination of axillary lymph nodes (ALN) in breast cancer patients is carried out physically and by ultrasound imaging; unsuspicious nodes will lead to a sentinel node (SN) procedure, suspicious ones require axillary dissection (AD). Pre-operative biopsy techniques like fine needle aspiration (FNA) or core biopsy (CB) may reduce the number of false "suspicious" cases and prevent overtreatment. We evaluated the effectiveness of both biopsy techniques. MATERIALS AND METHODS: After physical and ultrasound examination 241 suspicious ALNs were found in 214 patients. Ultrasound-guided FNA and/or CB procedures were chosen randomly, resulting in 138 FNA and 86 CB. In 17 further events both FNA and CB were employed. The samples were examined in our Cytology lab or in the Pathology Department and the findings correlated with post-operative histological lymph node reports. Patients with histologically proven breast cancer underwent sentinel node biopsy, cytologically or histologically positive FNA/CB-findings prompted ALN dissection. RESULTS: Out of 155 FNA samples 34 were not representative (21.9%), 89 showed no tumor cells (57.4%), 30 showed positive tumor cells (19.4%), leaving two missing. All 103 CB showed representative material, positive in 62 (60.2%) and negative in 41 (39.8%) cases. Correlation with histological reports revealed a statistically non-significant advantage for CB over FNA regarding total accuracy (92.9% vs. 78.3%) and sensitivity (92% vs. 73.7%). CONCLUSIONS: Preoperative CB and alternative FNA are valuable complementary methods of predicting ALN involvement in breast cancer patients and may spare the patient unnecessary ALN dissection.

Identification of 3q oncogene SEC62 as a marker for distant metastasis and poor clinical outcome in invasive ductal breast cancer.

PURPOSE: In previous studies, we have shown that SEC62 has an essential function in cell migration, epithelial-to-mesenchymal transition, and endoplasmic reticulum stress tolerance of cancer cells. SEC62 expression correlated with distant and lymph node metastasis and poor outcome in different cancer entities. In this initial study, we investigated SEC62 expression and its possible role as a prognostic and predictive biomarker in breast cancer (BC). METHODS: Formalin-fixed, paraffin-embedded tissue samples of 53 BC patients were analyzed by immunohistochemistry. The immunoreactive score (IRS) according to Remmele and Stegner was evaluated and correlated with clinico-pathological findings and overall survival (OS). RESULTS: We found increased SEC62 protein levels in tumor tissue compared to tumor-free tissue samples from the same patients. Tumors with high SEC62 expression (IRS > 8), or containing isolated cells with high SEC62 staining intensity, independent of the IRS, had more frequently distant metastases (48.4% vs. 18.2%; p = 0.024 and 47.4 vs. 6.7%; p = 0.005, respectively). Overall survival was significantly worse in BC patients with high SEC62 expression (SEC62 IRS > 8) (54.8% vs. 81.8%; p = 0.011) and in cases with isolated high-intensity SEC62 staining cells independently of SEC62 IRS (55.3% vs 93.3%; p = 0.024). CONCLUSIONS: We are the first to describe the SEC62 expression and its correlation to clinicopathological parameters in mammary carcinoma. Our results suggest that SEC62 expression may serve as a prognostic marker for patients with invasive ductal breast cancer.

Sec62/Ki67 and p16/Ki67 dual-staining immunocytochemistry in vulvar cytology for the identification of vulvar intraepithelial neoplasia and vulvar cancer: a pilot study.

PURPOSE: The aim of this study was to analyze the diagnostic performance of a newly established immunocytochemical dual-staining protocol for the simultaneous expression of SEC62 and Ki67 in vulvar liquid-based cytology specimens for the identification of vulvar intraepithelial neoplasia (VIN) and vulvar cancer. In addition, we investigated the p16/Ki67 dual stain, which has already been established in cervical cytology. MATERIALS AND METHODS: For this pilot study, residual material from liquid-based cytology was collected retrospectively from 45 women. The presence of one or more double-immunoreactive cells was considered as a positive test result for Sec62/Ki67 and p16/Ki67 dual staining. The test results were correlated with the course of histology. RESULTS: All cases of VIN and vulvar cancer were Sec62/Ki67 and p16/Ki67 dual-stain positive, and normal and low-grade squamous intraepithelial lesions were all negative. The sensitivity of cytology for VIN + cases was 100% (22/22), whereas punch biopsy classified one case of vulvar carcinoma as inflammation. All cases with high-intensity (grades 3 and 4) Sec62 staining in Sec62/Ki67-positive cases were carcinomas. CONCLUSIONS: The results of this study demonstrate that Sec62/Ki67 and p16 Ki67 dual-staining cytology could be a promising adjunctive diagnostic tool for VIN and squamous cell carcinoma, in addition to standard histology.

Ocular changes in nephropathic cystinosis: The course of the gold-dust.

PURPOSE: Cystinosis is an autosomal recessive inherited lysosomal storage disease with an incidence of 1:100.000 up to 1:200.000 caused by a gene mutation of a lysosomal transport protein resulting in deposition of cystine in lysosomes in all cells and tissues. In the cornea, crystalline, gold-dust deposition of cystine leads to visual impairment, recurrent erosions, photophobia, epiphora and blepharospasmus. Standard therapy is topical and systemic application of cysteamine which may resolve the accumulated cystine crystals. PATIENT AND METHODS: This is a case report of a thirty-one-year-old patient who already underwent renal transplantation because of nephropathic cystinosis. Visual impairment by cystine crystal deposition was aggravated by a central avascular pannus formation in his right eye. Penetrating keratoplasty was performed in intention to improve the patient's visual acuity and life quality. RESULTS: After penetrating keratoplasty in the right eye, there was only a slight visual improvement. OCT scans of the macula revealed intraretinal cystine crystals and a cystoid macular edema, which was treated with a bevacizumab injection. Transmission electron microscopy of the excised cornea revealed spiky intracorneal inclusions and confocal in vivo microscopy of the left eye allowed detailed visualization of the cystine crystal deposition. CONCLUSIONS: There is a variability of ocular manifestations of nephropathic cystinosis. Ophthalmologists have a central role in the early diagnosis of cystinosis as mostly the first manifestation are cystine crystals in the cornea. Penetrating keratoplasty may be one of the therapeutical options. Nevertheless, the patient has to be informed about the limited prognosis because of the persisting underlying disease.

A novel mouse model of human prostate cancer to study intraprostatic tumor growth and the development of lymph node metastases.

BACKGROUND: In this study, we aimed to establish a versatile in vivo model of prostate cancer, which adequately mimics intraprostatic tumor growth, and the natural routes of metastatic spread. In addition, we analyzed the capability of high-resolution ultrasonography (hrUS), in vivo micro-CT (µCT), and 9.4T MRI to monitor tumor growth and the development of lymph node metastases. METHODS: A total of 5 × 105 VCaP cells or 5 × 105 cells of LuCaP136- or LuCaP147 spheroids were injected into the prostate of male CB17-SCID mice (n = 8 for each cell type). During 12 weeks of follow-up, orthotopic tumor growth, and metastatic spread were monitored by repetitive serum-PSA measurements and imaging studies including hrUS, µCT, and 9.4T MRI. At autopsy, primary tumors and metastases were harvested and examined by histology and immunohistochemistry (CK5, CK8, AMACR, AR, Ki67, ERG, and PSA). From imaging results and PSA-measurements, tumor volume doubling time, tumor-specific growth rate, and PSA-density were calculated. RESULTS: All 24 mice developed orthotopic tumors. The tumor growth could be reliably monitored by a combination of hrUS, µCT, MRI, and serum-PSA measurements. In most animals, lymph node metastases could be detected after 12 weeks, which could also be well visualized by hrUS, and MRI. Immunohistochemistry showed positive signals for CK8, AMACR, and AR in all xenograft types. CK5 was negative in VCaP- and focally positive in LuCaP136- and LuCaP147-xenografts. ERG was positive in VCaP- and negative in LuCaP136- and LuCaP147-xenografts. Tumor volume doubling times and tumor-specific growth rates were 21.2 days and 3.9 %/day for VCaP-, 27.6 days and 3.1 %/day for LuCaP136- and 16.2 days and 4.5 %/day for LuCaP147-xenografts, respectively. PSA-densities were 433.9 ng/mL per milliliter tumor for VCaP-, 6.5 ng/mL per milliliter tumor for LuCaP136-, and 11.2 ng/mL per milliliter tumor for LuCaP147-xenografts. CONCLUSIONS: By using different monolayer and 3D spheroid cell cultures in an orthotopic xenograft model, we established an innovative, versatile in vivo model of prostate cancer, which enables the study of both intraprostatic tumor growth as well as metastatic spread to regional lymph nodes. HrUS and MRI are feasible tools to monitor tumor growth and the development of lymph node metastases while these cannot be visualized by µCT.

Nontypeable Haemophilus influenzae-Promoted Proliferation of Kras-Induced Early Adenomatous Lesions Is Completely Dependent on Toll-Like Receptor Signaling.

Chronic obstructive pulmonary disease (COPD) is a risk factor for lung cancer. COPD is characterized by chronic airway inflammation and lung infections. The airways of patients with COPD are frequently colonized with bacteria [eg, nontypeable Haemophilus influenzae (NTHi)] that cause pulmonary inflammation and exacerbations. Pulmonary adenocarcinomas are frequently associated with an activating mutation in the KRAS gene. We determined the function of Toll-like receptor (TLR) signaling on the progression of Kras-induced early adenomatous lesions in the lung. Wild-type (WT) mice and mice doubly deficient in Tlr-2 and -4 (Tlr2/4-/-), both with an oncogenic Kras allele in lung epithelium, were exposed to NTHi for 4 weeks. Exposure to NTHi resulted in increased tumor proliferation and growth in WT mice, but not in Tlr2/4-/- mice. Alveolar adenomatous hyperplasia and adenocarcinoma were significantly increased in WT mice compared with Tlr2/4-/- mice. The average size of tumors was significantly larger in WT mice, whereas there was no difference in the number of alveolar lesions between WT and Tlr2/4-/- mice. NTHi-induced pulmonary neutrophilic inflammation and tumor-associated neutrophils were reduced in Tlr2/4-/- mice. Thus, subsequent to a driver mutation, NTHi-induced inflammation promotes proliferation of early adenomatous lesions in a TLR-dependent manner.