Experience with telepathology in combination with diagnostic assistance systems in countries with restricted resources.

INTRODUCTION: We describe the use of telepathology in countries with restricted resources using two diagnosis assistance systems (Isabel and Memem7) in addition to the diagnoses made by experts in pathology via the iPath-Network. METHODS: A total of 156 cases, largely from Afghanistan, were analysed; 18 cases had to be excluded because of poor image quality. RESULTS: Of the remaining 138 cases (100%), a responsible physician provided a tentative diagnosis for 61.6% of them. With a diagnosis from a consultant pathologist, it was then possible to make a definite diagnosis in 84.8% of cases on the basis of images taken from hematoxylin and eosin staining sections alone. The use of the diagnosis assistance systems resulted in an ordered list of differential diagnoses in 82.6% (IsabelHealth) and in 74.6% (Memem7) of cases, respectively. Adding morphological terminology reduced the list of possible diagnoses to 52.2% (72 cases, Memem7), but improved their quality. DISCUSSION: In summary, diagnosis assistance systems are promising approaches to provide physicians in countries with restricted resources with lists of probable differential diagnoses, thus increasing the plausibility of the diagnosis of the consultant pathologist.

[Lethal intravenous injection of benzine]. / Intravenöse Injektion von Waschbenzin mit tödlichem Verlauf.

A man who suffered from chronic pain syndrome died two days after intravenous injection of 2 ml benzine. Previous suicide attempts by drug intoxication and strangulation had failed. Death occurred due to multi-organ failure. We present the results of the clinical, morphological and toxicological examinations performed.

Benign cystic mesothelioma of the peritoneum in a 12-year-old boy, diagnosed via telepathology.

Low resource countries often have inadequate healthcare systems that, among other problems, suffer from a severe shortage of local expertise. In fields such as pathology, however, where diagnoses are mainly based on macroscopic and histological images, telemedicine can provide an opportunity to supplement the local capabilities by involving a world-wide network of experts. For this, the local pathologist can upload images to an online platform and then consult with international colleagues via teleconferencing, which can be particularly useful in rare or difficult cases. We present the case of a 12-year-old Afghan boy with benign cystic mesothelioma, an extremely rare benign tumour. Using the online platform IPath, we were able to diagnose the eighth reported case of this rare condition in a child, in collaboration with our colleagues in Afghanistan.

FOXE1 association with differentiated thyroid cancer and its progression.

BACKGROUND: Single nucleotide polymorphisms (SNPs) near thyroid transcription factor genes (FOXE1 rs965513/NKX2-1 rs944289) have been shown to be associated with differentiated thyroid cancer (DTC) in Caucasoid populations. We investigated the role of those SNPs in German patients with DTC and also extended our analysis to tumor stages and lymphocytic infiltration of the tumors (ITL). METHODS: Patients with DTC (n=243; papillary, PTC; follicular, FTC) and healthy controls (HC; n=270) were analyzed for the rs965513 and rs944289 SNPs. RESULTS: The case-control analysis for rs965513 SNP showed that the genotypes "AA," "AG," and minor allele "A" were more frequent in patients with DTC than in HC (pronounced in PTC p(genotype)=0.000084, p(allele)=0.006 than FTC p(genotype)=0.29 and p(allele)=0.06). Furthermore, subgroup analysis of the DTC patients stratified for primary tumor stage (T1-T2, T3-T4), the absence or presence of regional lymph node metastases (N0, N1), for distant metastases (M0, M1), as well as for ITL, showed an association of rs965513 with stages T1-T2, T1-T3, N1, and absence of ITL. The NKX2-1 SNP rs944289, however, was not associated with DTC. CONCLUSION: Our results confirm that the FOXE1 rs965513 SNP confers an increased risk for DTC in the German population, particularly allele "A" and the genotypes "AA" and "AG" for PTC. This increased risk was also observed in advanced tumor stages and absence of ITL, which may reflect the course of a more aggressive disease. The NKX2-1 rs944289 SNP, however, appears to play a secondary role in the development of DTC in the German population.

A hybrid approach to telepathology in Cambodia.

We established a hybrid telepathology network at the Children's Surgical Centre (CSC) in Cambodia, based on store-and-forward communication using iPATH and videoconferencing using Skype. We retrospectively analysed all data from the CSC stored on the iPATH server and reviewed the patient notes over an 8-month period. Of 115 patients for histopathology diagnosis during the study period, 38 cases were uploaded onto iPATH for further telemedicine discussion. The median number of days it took a specialist, other than the local one, to comment on the case on iPATH was 5 days (range 0-15). In three cases (8%) there was no reply from a specialist on iPATH. During the study period, seven clinical conferences were held, with an average of 6 cases (range 4-7) discussed at each conference. All 38 cases discussed had a final agreed diagnosis and firm management plans were made. Of the 24 cases where proactive management was advised, 17 patients followed through with the recommendations. Although the combination of video consultations and store-and-forward communication has not been used much before in the developing world, it has benefited patient care and outcomes at the CSC.

Impaired vitamin D activation and association with CYP24A1 haplotypes in differentiated thyroid carcinoma.

BACKGROUND: Common polymorphisms of the vitamin D receptor gene have been reported to affect the risk of breast, colon, prostate, and differentiated thyroid cancer (DTC), but polymorphisms within the genes of vitamin D metabolizing enzymes have not been studied in DTC. The aim of the present study was to investigate the genes for vitamin D enzymes in patients with DTC and healthy controls (HC) as well as the vitamin D (25-hydroxyvitamin D(3), and 1,25-hydroxyvitamin) status. METHODS: German patients (n=253) with DTC (papillary thyroid carcinoma [PTC] and follicular thyroid carcinoma [FTC]) and HC (n=302) were genotyped for polymorphisms within the vitamin D metabolizing enzymes such as 25-hydroxylase (CYP2R1[rs12794714, rs10741657]), 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1[rs10877012, rs4646536]), and 25-hydroxyvitamin D 24-hydrolase (CYP24A1[rs927650, rs2248137, rs2296241]). Furthermore, the 25-hydroxyvitamin D(3) [25(OH)D(3)] and 1,25-hydroxyvitamin [1,25(OH)(2)D(3)] plasma levels were measured by a radioimmunoassay. RESULTS: There was no difference in the genotypes; however, the CYP24A1 haplotype analysis showed that rs2248137C/rs2296241A (13.1% vs. 19.1%; corrected p [pc]=0.04) was less frequent in the PTC, whereas the haplotypes rs2248137C/rs2296241G (56.0% vs. 41.9%; pc=0.03), rs927650C/rs2296241G (22.5% vs. 8.4%; pc=1.6×10(-3)), and rs927650C/rs2248137C/rs2296241G (21.1% vs. 7.3%; pc=1.5×10(-3)) were more frequent in the FTC compared with HC. Furthermore, if patients and controls were grouped according to four 25(OH)D(3) categories (severely deficient, deficient, insufficient, and sufficient), then the patients with both DTC subtypes had significantly lower levels of circulating 1,25(OH)(2)D(3), especially in the group with a deficient 25(OH)D(3) status compared with the controls. Although the polymorphisms showed no differences stratified for the four 25(OH)D(3) categories, the activation status by 1,25(OH)(2)D(3) differed significantly depending on the genotypes of the investigated CYP24A1 polymorphisms. CONCLUSIONS: A higher risk for DTC is conferred by haplotypes within the CYP24A1 gene, low circulating 25(OH)D(3) levels (deficiency), and a reduced conversion to 1,25(OH)(2)D(3). These results confirm and extend previous observations and also support a role of the vitamin D system in the pathogenesis of DTC. How deficient 25(OH)D(3) levels in combination with certain CYP24A1 haplotypes affect vitamin D activation is the subject of future studies.

Chemotherapy-associated angiogenesis in neuroblastoma tumors.

The influences of cytotoxic drugs on endothelial cells remain incompletely understood. Herein, we examined the effects of chemotherapeutic agents in experimental angiogenesis models and analyzed vessel densities in clinical neuroblastoma tumor samples. Cisplatin (20 to 500 ng/mL), doxorubicin (4 to 100 ng/mL), and vincristine (0.5 to 4 ng/mL), drugs commonly involved in neuroblastoma therapy protocols, induced pro-angiogenic effects in different angiogenesis models. They enhanced endothelial cell tube formation, endothelial cell sprouting from spheroids, formation of tip cells in the sprouting assay, expression of αvß3 integrin, and vitronectin binding. All three drugs increased global cellular kinase phosphorylation levels, including the angiogenesis-relevant molecules protein kinase Cß and Akt. Pharmacological inhibition of protein kinase Cß or Akt upstream of phosphatidylinositol 3-kinase reduced chemotherapy-induced endothelial cell tube formation. Moreover, the investigated chemotherapeutics dose dependently induced vessel formation in the chick chorioallantoic membrane assay. Tumor samples from seven high-risk patients with neuroblastoma were analyzed for vessel density by IHC. Results revealed that neuroblastoma samples taken after chemotherapy consistently showed an enhanced microvessel density compared with the corresponding samples taken before chemotherapy. In conclusion, our data show that chemotherapy can activate endothelial cells by inducing multiple pro-angiogenic signaling pathways and exert pro-angiogenic effects in vitro and in vivo. Moreover, we report a previously unrecognized clinical phenomenon that might, in part, be explained by our experimental observations: chemotherapy-associated enhanced vessel formation in tumors from patients with neuroblastoma.

Chemoresistance acquisition induces a global shift of expression of aniogenesis-associated genes and increased pro-angogenic activity in neuroblastoma cells.

BACKGROUND: Chemoresistance acquisition may influence cancer cell biology. Here, bioinformatics analysis of gene expression data was used to identify chemoresistance-associated changes in neuroblastoma biology. RESULTS: Bioinformatics analysis of gene expression data revealed that expression of angiogenesis-associated genes significantly differs between chemosensitive and chemoresistant neuroblastoma cells. A subsequent systematic analysis of a panel of 14 chemosensitive and chemoresistant neuroblastoma cell lines in vitro and in animal experiments indicated a consistent shift to a more pro-angiogenic phenotype in chemoresistant neuroblastoma cells. The molecular mechanisms underlying increased pro-angiogenic activity of neuroblastoma cells are individual and differ between the investigated chemoresistant cell lines. Treatment of animals carrying doxorubicin-resistant neuroblastoma xenografts with doxorubicin, a cytotoxic drug known to exert anti-angiogenic activity, resulted in decreased tumour vessel formation and growth indicating chemoresistance-associated enhanced pro-angiogenic activity to be relevant for tumour progression and to represent a potential therapeutic target. CONCLUSION: A bioinformatics approach allowed to identify a relevant chemoresistance-associated shift in neuroblastoma cell biology. The chemoresistance-associated enhanced pro-angiogenic activity observed in neuroblastoma cells is relevant for tumour progression and represents a potential therapeutic target.

Vitamin D receptor polymorphisms in differentiated thyroid carcinoma.

BACKGROUND: Vitamin D receptor (VDR) expression has been shown to be upregulated in several tumors and is supposed to represent an important endogenous response to tumor progression. To investigate the role of the VDR gene and its influence on 25(OH)D(3) and 1,25(OH)(2)D(3) plasma levels in thyroid carcinoma, we analyzed four VDR polymorphisms in patients and healthy controls (HC). METHODS: Patients with thyroid carcinoma (n = 172) (n = 132 for papillary and n = 40 for follicular) and HC (n = 321) were genotyped for the ApaI (rs7975232), TaqI (rs731236), BsmI (rs1544410), and FokI (rs10735810) polymorphisms within the VDR gene and correlated with 25(OH)D(3) and 1,25(OH)(2)D(3) plasma levels. RESULTS: The genotypes AA of the ApaI (rs7975232) and FF of the FokI (rs10735810) polymorphisms were significantly less frequent (12.5% vs. 35.2% and 25% vs. 42.1%, respectively, both corrected p [p(c)] = 0.04) in patients with follicular thyroid cancer (FTC) than in HC. Additionally, the haplotypes, Ta (57.5% vs. 41.4%; p(c) = 0.0207), af (24.6% vs. 14.3%; p(c) = 0.0116), Tab (51.1% vs. 36.8%; p(c) = 0.0495), and Tabf (18.7% vs. 13.6%; p(c) = 0.0240) were more frequent, whereas the haplotypes AF (17.1% vs. 37.2%; p(c) = 0.0008), BF (11.4% vs. 31.9%; p(c) = 0.012), tF (7.9% vs. 25.5%; p(c) = 0.0016), and tABF (7.6% vs. 23%; p(c) = 0.0115) were less frequent in the FTC patients compared to HC. Neither genotype nor haplotype frequencies differed between patients with papillary thyroid cancer (PTC) and HC. Further, individuals with PTC and FTC had a significantly lower level of circulating 1,25(OH)(2)D(3) compared to controls. In contrast, no differences of the 25(OH)D(3) concentration between patients and HC were observed. VDR polymorphisms were not associated with 25(OH)D(3) and 1,25(OH)(2)D(3) plasma levels. CONCLUSIONS: Lower circulating levels of 1,25(OH)(2)D(3) are observed in patients with differentiated thyroid carcinoma. Further, while the alleles AA and FF of the ApaI (rs7975232) and FokI (rs10735810) VDR polymorphisms and the haplotype tABF confer to protection from follicular carcinoma, the haplotype Tabf appeared to be associated with an increased FTC risk. Since this is the first report associating VDR polymorphisms with thyroid carcinoma, these findings need to be confirmed in studies with larger numbers of patients.

QPRT: a potential marker for follicular thyroid carcinoma including minimal invasive variant; a gene expression, RNA and immunohistochemical study.

BACKGROUND: The differential diagnosis between follicular thyroid adenoma and minimal invasive follicular thyroid carcinoma is often difficult for several reasons. One major aspect is the lack of typical cytological criteria in well differentiated specimens. New marker molecules, shown by poly- or monoclonal antibodies proved helpful. METHODS: We performed global gene expression analysis of 12 follicular thyroid tumours (4 follicular adenomas, 4 minimal invasive follicular carcinomas and 4 widely invasive follicular carcinomas), followed by immunohistochemical staining of 149 cases. The specificity of the antibody was validated by western blot analysis RESULTS: In gene expression analysis QPRT was detected as differently expressed between follicular thyroid adenoma and follicular thyroid carcinoma. QPRT protein could be detected by immunohistochemistry in 65% of follicular thyroid carcinomas including minimal invasive variant and only 22% of follicular adenomas. CONCLUSION: Consequently, QPRT is a potential new marker for the immunohistochemical screening of follicular thyroid nodules.

Cisplatin-resistant neuroblastoma cells express enhanced levels of epidermal growth factor receptor (EGFR) and are sensitive to treatment with EGFR-specific toxins.

PURPOSE: Neuroblastomas frequently show expression of the epidermal growth factor receptor (EGFR) and may therefore be susceptible to EGFR-targeted therapies. Here, EGFR expression and functionality was investigated in parental chemosensitive neuroblastoma cell lines (UKF-NB-3, IMR-32, NLF, SH-SY5Y) and their cisplatin-resistant sublines (UKF-NB-3(r)CDDP(1000), IMR-32(r)CDDP(1000), NLF(r)CDDP(1000), and SH-SY5Y(r)CDDP(500)). Moreover, the EGFR antibody cetuximab, the EGFR tyrosine kinase inhibitor Tyrphostin B46, and recombinant EGFR-targeted toxins were investigated for their influence on the viability and growth of neuroblastoma cells. EXPERIMENTAL DESIGN: EGFR expression and function was measured by flow cytometry or Western blot. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was examined by immunostaining for active caspase-3 or cleaved poly(ADP-ribose) polymerase. Cellular binding of FITC-labeled immunotoxins was studied by flow cytometry, and cellular uptake was studied by confocal laser scanning microscopy. RESULTS: The EGFR-targeted antibody and growth factor toxins scFv(14E1)- Pseudomonas exotoxin A (ETA) and TGF-alpha-ETA exerted anti-cancer effects in neuroblastoma cell lines that were insensitive to cetuximab or EGFR tyrosine kinase inhibitors. Furthermore, adaptation of chemosensitive neuroblastoma cells to cisplatin increased EGFR expression and sensitivity to both recombinant toxins. Treatment of chemosensitive neuroblastoma cells with cisplatin reversibly increased EGFR expression, whereas cisplatin-resistant cells showed enhanced EGFR expression independent of the presence of cisplatin. Combination treatment with scFv(14E1)-ETA or TGF-alpha-ETA and cisplatin exerted significantly improved anticancer effects compared with either single treatment in parental neuroblastoma cells, cisplatin-resistant sublines, and primary cultures. CONCLUSIONS: EGFR-targeted cytotoxic reagents such as scFv(14E1)-ETA and TGF-alpha-ETA represent promising candidates for further development as antineuroblastoma agents, especially in combination with cisplatin.

Fatal haemorrhage due to extensive fragility of medium- and large-sized arteries and veins in a young patient with neurofibromatosis 1.

BACKGROUND: Neurofibromatosis type 1 (NF1) is one of the most frequently inherited diseases. Vascular manifestations have been reported, mostly concerning stenosis of the renal artery or spontaneous rupture of single arteries. Also, venous aneurysms have been reported. METHODS: We present clinical and pathological findings of a young patient with NF1 with lethal bleeding due to spontaneous rupture of a lumbal and renal artery. RESULTS: High fragility of several arteries as well as veins in the abdominal and pelvic arterial and venous system resulted in a destroyed vessel structure caused by invading neurogen fibres. CONCLUSIONS: In rare cases, NF1 is associated with a severe systemic vasculopathy concerning the arterial and venous vascular system.

The aberrant coexpression of several receptor tyrosine kinases is largely restricted to EBV-negative cases of classical Hodgkin's lymphoma.

The Hodgkin-Reed/Sternberg (HRS) cells of classical Hodgkin's lymphoma (HL) aberrantly express up to 7 different receptor tyrosine kinases (RTK) with extensive heterogeneity regarding the number and combinations of expressed RTKs in individual cases and a more prominent coexpression in nodular-sclerosis (ns) than mixed-cellularity (mc) HL. To investigate whether RTK expression patterns are related to other pathogenetic mechanisms and clinical behaviour, we analysed a large collection of EBV(+) and EBV(-) cases of ns and mc subtype and cases with relapses for expression of the 7 RTKs. No specific relation of any RTK to a specific group of cases was observed. The analysis of average numbers of expressed RTKs per case as a measure for strength of overall RTK signalling revealed a relation with the histological subtype and the EBV-status. RTK coexpression was significantly higher in EBV(-) nsHL cases compared to both EBV(-) and EBV(+) mcHL cases. Among mcHL cases RTK coexpression was significantly higher in EBV(-) compared to EBV(+) cases. Coexpression of 3 and more RTKs was largely restricted to EBV(-) cases. The inverse correlation between strong RTK signalling and presence of EBV may indicate that RTK signalling can at least partially replace the role of EBV in HRS cell pathogenesis. For cases with aberrant coexpression of several RTKs inclusion of RTK inhibitors in therapy regimens may be a novel option.

[Global gene expression analysis and novel signalling pathways in Hodgkin lymphoma]. / Globale Genexpressionsanalysen und neue Signalwege beim Hodgkin Lymphom.

To identify pathogenetic mechanisms in Hodgkin lymphoma (HL) we performed global gene expression profiling of four HL derived cell lines and compared the expression profiles with those of normal B cells and B cell non-HL. This analysis revealed a global loss of B-cell specific gene expression in Hodgkin-Reed/Sternberg (HRS) cells (21). Further analysis showed that ABF-1 and Id2, which are both negative regulators of the B cell master transcription factor E2A and likely also Pax-5, are aberrantly expressed in HRS cell lines (11, 17). For Id2, immunohistochemistry showed expression in the HRS cells of all cases, and E2A could be coimmunoprecipitated with Id2 from HRS cell lines, indicating that the aberrant Id2 expression may indeed contribute to the loss of the B-cell specific gene expression in HL (17). An analysis of the global gene expression data for aberrant expression of genes which are frequently involved in neoplastic transformation identified six receptor tyrosine kinases (RTK) aberrantly expressed in HRS cell lines. All RTKs were also (co)-expressed in primary cases and mostly activated by auto- or paracrine mechanisms (18). Analysis of greater number of cases identified a subgroup of HL which encompasses about 20 % of cases characterised by coexpression of at least four of the RTKs. An survey of several B cell lymphoma types with a pan-phospho-tyrosine specific antibody indicated that mediastinal large B-cell lymphoma is beside HL the only entity where aberrant TK activities cause an aberrantly high cellular phospho-tyrosine content in a significant fraction of cases.