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We described the case of a peripancreatic paraganglioma (PGL) misdiagnosed as pancreatic lesion. Surgical exploration revealed an unremarkable pancreas and a large well-defined cystic mass originating at the mesocolon root. Radical enucleation of the mass was performed, preserving the pancreatic tail. Histologically, a diagnosis of PGL was rendered. Interestingly, two previously unreported mutations, one affecting the

Introduction. Published evidence suggests that immunonutrition has the potential to decrease postoperative complications and reduce length of stay in patients undergoing surgery for colorectal cancer. However, only a few studies have analyzed the effects of immunonutrition on tumor microenvironment and evaluated its prognostic impact. Material and methods. This is a single center retrospective study enrolling 50 patients undergoing elective surgery for colorectal cancer managed with immunonutrition and 50 patients managed with standard nutrition for comparison. Tumor microenvironment was analyzed before (on the biopsy at the time of diagnosis) and after (on the matched surgical specimen) administration of immunonutrition. Immune function related indicators, including cytotoxic T-lymphocytes, helper T-cells, antigen presenting cells, natural killer cells, T-exhausted lymphocytes, T-regulatory cells, M1 and M2 tumor associated macrophages and PD-L1 expression were assessed by immunohistochemistry. For both groups, clinicopathological data were collected and a 5-year follow-up was available. Results. We found that immunonutrition significantly activated the T-cell response against cancer, alter tumor microenvironment phenotype towards M2 polarization and inhibits the PD1/PD-L1 axis. A lower rate of postoperative complications and a shorter length of stay (p = 0.04) were observed in the immune nutrition group. Compared to standard nutrition group, patients managed wit immune nutrition showed a higher 5-year overall survival (p = 0.001). Finally, immune nutrition allowed to reduce the hospital care costs. Conclusions. Immunonutrition modulates tumor microenvironment by improving immune function and could prolong survival in patients undergoing elective surgery for colorectal cancer. Further studies are needed to optimize IN protocols and confirm their prognostic impact.

Introduction: Pulmonary carcinosarcoma is a rare histological subtype of non-small cell lung cancer, defined by the combination of epithelial and mesenchimal elements. Prognosis is usually dismal, with a median survival of about 6 months. The use of immunotherapy by blockade of PD1/PD-L1 immune checkpoint signaling has been shown to improve patients’ survival. However, local aggressiveness and distant metastases are frequent. Spread to the gastrointestinal tract is seldom reported. The genetic landscape of the disease has only recently begun to emerge, pointing at TP53, KRAS, EGFR and MET as the most common mutated genes. Case description: We describe the case of a metastatic patient with 37 months overall survival, treated by an aggressive multimodal approach combining surgery, chemotherapy, radiotherapy and immunotherapy. To shed new light on the molecular basis for sarcomatoid component in lung carcinoma, we performed next generation sequencing analysis of the squamous and sarcomatoid component by the two sites. We demonstrated a clonal origin and hypermutability of the sarcomatous elements that may account for the good response to immunotherapy. Moreover, we identified some mutations involving TP53 and EGFR genes, targetable by already available drugs. Conclusions: We depicted a model of how a squamous cell carcinoma can differentiate during its natural history into sub-clonal populations with different features and may ultimately result in a neoplasm (i.e. pulmonary carcinosarcoma) showing clonal heterogeneity. Our data might contribute to a better understanding of the pathogenesis and molecular mechanisms of this rare tumor and open new ways for a more tailored approach.

The downstaging of gastric cancer has recently gained particular attention in the field of gastric cancer surgery. The phenomenon is mainly due to an inappropriate sampling of lymph nodes during standard lymphadenectomy. Hence, collection of the maximum number of lymph nodes is a critical factor affecting the outcome of patients. None of the techniques proposed so far have demonstrated a real efficiency in increasing the number of identified lymph nodes. To harvest the maximum number of lymph nodes, we designed a protocol for on-site macroscopic evaluation and sampling of lymph nodes according to the Japanese Gastric Cancer Association protocol. The procedure was carried out by a surgeon/pathologist team in the operating room. We enrolled one hundred patients, 50 of whom belonged to the study group and 50 to a control group. The study group included patients who underwent lymph node dissection following the proposed protocol; the control group encompassed patients undergoing standard procedures for sampling. We compared the number and maximum diameter of lymph nodes collected in both groups, as well as some postoperative variables, the 30-day mortality and the overall survival. In the study group, the mean number of lymph nodes harvested was higher than the control one (p = 0.001). Moreover, by applying the proposed technique, we sampled lymph nodes with a very small diameter, some of which were metastatic. Noticeably, no difference in terms of postoperative course was identified between the two groups, again supporting the feasibility of an extended lymphadenectomy. By comparing the prognosis of patients, a better overall survival (p = 0.03) was detected in the study group; however, to date, no long-term follow-up is available. Interestingly, patients with metastasis in node stations number 8, 9, 11 or with skip metastasis, experienced a worse outcome and died. Based on our preliminary results, the pathologist/surgeon team approach seems to be a reliable option, despite of a slight increase in sfaff workload and technical cost. It allows for the harvesting of a larger number of lymph nodes and improves the outcome of the patients thanks to more precise staging and therapy. Nevertheless, since a higher number of patients are necessary to confirm our findings and assess the impact of this technique on oncological outcome, our study could serve as a proof-of-concept for a larger, multicentric collaboration.

Background/Aims Gastrointestinal stromal tumors (GISTs) strongly express a receptor tyrosine kinase (RTK, c-KIT-CD117) harboring a KIT mutation that causes constitutive receptor activation leading to the development and growth of tumors; 35% of GISTs without KIT mutations have platelet-derived growth factor receptor alpha (PDGFRA) mutations, and the type of mutation plays an important role in the response to treatment. This study aimed to establish the frequency of stop codon mutations in the RTKs, KIT, and PDGFRA, in GISTs and correlate this molecular alteration with protein expression and treatment responsiveness. Methods Seventy-nine GISTs were analyzed for both KIT and PDGFRA mutations. Immunohistochemical expression was studied in tissue microarray blocks. Results We found three rare KIT mutations in exon 11 that induced a stop codon, two at position 563 and one at position 589, which have never been described before. All three tumors were CD117-, DOG1-, and CD34-positive. Two patients with a KIT stop codon mutation did not respond to imatinib therapy and died shortly after treatment. Conclusions The association between stop codon mutations in KIT and patient survival, if confirmed in a larger population, may be useful in choosing effective therapies.

Objective: To examine the seroprevalence, correlates and characteristics of Celiac disease (CD) in a population sample of a Northern Central Area of Italy, by a monocenter retrospective study. Methods: Between 2006 and 2010, serum samples of 9371 subjects (age range 6 months to 91 years) were screened for tissue transglutaminase IgA antibodies (IgA-tTG) by the Immunologia-Allergologia Unit of AUSL1 Massa-Carrara, an area with a population of approximately 150,000. Endomysial IgA antibodies (EMA), HLA typing and small-bowel biopsy were also performed when indicated. Results: Of the 9371 subjects, 269 (2.87%) had positive antibody tests. The population was divided into several age groups and the highest prevalence (5.63%) was found in the 6 months-3 years group. The prevalence of IgA-tTG positivity was double in females compared to males. All IgA-tTG positive patients that were genotyped carried HLA-DQ2 or DQ8, none was a5 positive only. In positive IgA-tTG sera, levels of IgA-tTG were significantly higher in EMA positive than in EMA negative sera (p in children and in adults. Ninety-five/269 IgA-tTG positive subjects underwent biopsy. IgA-tTG levels were different according to the histological degree of the lesion. When EMA were evaluated in IgA-tTG positive subjects the number of EMA negative sera was significantly higher in adults than in children. Conclusions: In summary, this study provides a monocenter retrospective analysis of serological, histological and genetic parameters of subjects with suspicion of CD in an area of Northern Central Italy from 2006 to 2010.

Xenon and other inhalational agents induce cell and organ effects through different and only partially elucidated molecular mechanisms. In this study, we explored the gene transcription consequences of xenon exposure compared with nitrogen or nitrous oxide exposure in rat brain. Seven-day-old Sprague Dawley rats (n=24, 8 for each group) were exposed for 120 minutes to 75% xenon and 25% oxygen, 75% nitrogen and 25% oxygen (air), or 75% nitrous oxide and 25% oxygen. Using suppression subtractive hybridization, relative real-time polymerase chain reaction, and northern blot analyses of on/off gene expression, we were able to identify a set of genes that are significantly up-regulated by xenon exposure. These genes may help explain some of the molecular mechanisms that account for the neuropreconditioning effects exerted by xenon relative to nitrous oxide and air.

Although most Leydig cell tumors are benign, radical orchiectomy is currently considered the standard therapy. We retrospectively analyzed the long-term followup of a series of patients with Leydig cell tumors electively treated with testis sparing surgery.Between November 1990 and December 2005, 17 consecutive patients with Leydig cell tumors underwent testis sparing surgery on an elective basis. Preoperative evaluation included physical examination, serum markers for germ cell tumors, scrotal ultrasound, abdominal computerized tomography, chest x-ray and hormonal profile if clinically required. Testis sparing surgery was performed via an inguinal approach with spermatic cord clamping. Frozen section examination was performed in all cases, revealing Leydig cell tumors. Followup consisted of physical examination, scrotal ultrasound, abdominal computerized tomography and chest x-ray every 6 months for the first 2 years, then annually. Tumor recurrence and survival were evaluated.Mean patient age was 41.6 years (range 28 to 55). Medical referral was prompted by symptoms/signs such as infertility, gynecomastia or self-palpation of scrotal mass in 11 patients (64.7%), while in the remaining 6 (35.3%) the lesions were incidentally diagnosed. Hormonal profile was performed in 9 patients, showing abnormalities in all. Mean tumor diameter was 13.4 mm (range 5 to 31). Definitive pathological examination confirmed benign Leydig cell tumor in all cases. After a mean followup of 91 months (range 12 to 192), neither local recurrence nor distant metastases have been detected and all patients are alive without evidence of disease.In patients with Leydig cell tumors testis sparing surgery with frozen section examination provides an excellent long-term oncological outcome.

The TNM classification has been revised for the 2002 edition of the UICC publication to better stratify patients with intracapsular renal cell carcinoma (RCC) but few studies have been published to date to validate this new classification. Moreover, additional prognostic factors seem to be necessary to improve the prediction of intracapsular tumor aggressiveness and the definition of patient subgroups at high risk for metastases. We report the long-term results of the new TNM scheme. We evaluated the impact of DNA content, S-phase and MIB-1 (Dako, Glostrup, Denmark) score.A total of 136 patients with intracapsular clear cell RCC and a mean followup of 74 months were reclassified. Tumor specific survival (TSS) was compared with nuclear grade (NG), DNA content and proliferative status (S-phase fraction and MIB-1 score).TSS was 92%, 81.1% and 40.1% for pT1a, pT1b and pT2, respectively (p0.05). TSS according to DNA ploidy status (diploid vs aneuploid) was pT1a-95.2% vs 68.6% (p0.05), pT1b-90% vs 46.7% (p0.05) and pT2-49.2% vs 25% (p not significant). DNA ploidy was also significantly associated with survival when adjusted for NG. There was no significant association between TSS and MIB-1 score or tumor S-phase fraction.The 2002 TNM classification is a useful prognostic factor for evaluating organ confined RCC of the clear cell subtype. Evaluation of the DNA content in clear cell RCC appears to significantly improve the predictive value of the TNM staging system, especially in the pT1a and pT1b categories. Fuhrman NG alone or combined should be routinely used in such patients.

Introduction. Breast papillary lesions represent a heterogeneous group of tumors ranging from benign to malignant, including several intermediate forms. Malignant papillary tumors are rare and their molecular characterization is still limited. A few studies pointed to the presence of specific genetic alterations that could be relevant both for diagnostic purposes and to elucidate tumour development and progression. In order to look into the issue, we compared LOH relative frequencies of four microsatellite markers located on chromosome 16 in a set of morphologically different papillary breast lesions. LOH at TP53 locus was also analyzed throughout lesions. Materials and methods. Fifteen cases were analyzed. Sections including a malignant papillary lesion, a benign lesion (when available), and normal breast tissue were selected. Fifteen malignant and twelve benign areas were microdissected using the Leica laser microdissection system (AS LMD). After DNA extraction samples were tested for the following markers: TP53, D16S423, D16S310, DS163210 and D16S476, and analyzed on ABI PRISM 3100 (Applied Biosystems, Foster city CA). Results. Fourteen malignant lesions and twelve paired benign areas appeared to be informative for at least one of the four markers on chromosome 16. In particular, LOH at loci 16p13 and 16q21 was detected in both benign and malignant lesions, whereas LOH at locus 16q23 was limited to malignant lesions. Nine malignant and seven benign lesions were informative for LOH at TP53 locus, that was found to be significantly associated (p=0.01) with the malignant phenotype. Conclusions. Our data suggest an involvement of chromosome 16 mutations in the early steps of breast papillary tumorigenesis. TP53 deletion and possibly LOH at 16q23 appear to play a role as progression factors, being they significantly associated with malignant transformation of breast papilloma.

Neuroblastic tumours are a group of paediatric cancers with marked morphological heterogeneity. Neuroblastoma (Schwannian stroma-poor) (NB-SP) is composed of undifferentiated neuroblasts. Ganglioneuroblastoma intermixed (Schwannian stroma-rich) (GNBi-SR) is predominantly composed of Schwannian stromal (SS) and neuroblastic (Nb) cells. There are contrasting reports suggesting that SS cells are non-neoplastic. In the present study, laser capture microdissection (LCM) was employed to isolate SS and Nb cells. Chromosome 1p36 deletion and MYCN gene amplification were found to be associated in two out of seven NB-SPs, whereas no abnormalities were observed in five GNBi-SRs. In some cases, loss of heterozygosity (LOH) at 1p36 loci was detected in Nb cells but not in the bulk tumour by LCM; furthermore, LOH was also identified in both SS and tumour tissue of a GNBi-SR. DNA gain and loss studied by comparative genomic hybridization were observed at several chromosome regions in NB-SP but in few regions of GNBi-SR. Finally, gene expression profiles studied using an oligo-microarray technique displayed two distinct signatures: in the first, 32 genes were expressed in NB-SP and in the second, 14 genes were expressed in GNBi-SR. The results show that NB-SP is composed of different morphologically indistinguishable malignant cell clones harbouring cryptic mutations that are detectable only after LCM. The degree of DNA imbalance is higher in NB-SP than in GNBi-SR. However, when the analysis of chromosome 1p36 is performed at the level of microdissection, LOH is also observed in SS cells. These data provide supportive evidence that SS cells have a less aggressive phenotype and play a role in tumour maturation.

The tp73 gene, a tp53 homologue, has been sub-regionally mapped at 1p36.3, a chromosomal region frequently deleted in neuroblastoma. Due to its chromosomal localization and to the mono-allelic expression observed in some neuroblastoma cell lines, it was proposed that tp73 might be involved in the pathogenesis of neuroblastoma. Functional assays have demonstrated that tp73 can inhibit cell proliferation and induce apoptosis. The role of this gene in tumorigenesis, however, is still unclear. We analyzed tp73 expression in 95 sporadic neuroblastoma samples by RT-PCR and we detected the tp73 transcript in 46 cases (48.4%), without significant correlation with age, clinical stage or 3-year overall survival. A genetic polymorphism in the 2nd exon of tp73 was utilized to identify the transcribed allele in tumor-cell samples. Expression from only one of the tp73 alleles was found in 13 out of 16 heterozygous tumors, while in 3 samples both alleles were present. Genotype analysis of 73 patients and 150 controls showed a significant deviation (p = 0.0308) from the Hardy-Weinberg equilibrium for a tp73 allele only among neuroblastoma patients. The absence of correlation between tp73 expression and clinical stage, age and survival suggests that this gene does not play an essential function in the clinical course of the disease. However, the distribution of genomic tp73 alleles in patients indicates that a role of this gene in the development of neuroblastoma cannot be completely ruled out. Int. J. Cancer (Pred. Oncol.) 84:365–369, 1999. © 1999 Wiley-Liss, Inc.

Familial neuroblastoma occurs rarely. We studied a family with three children; one of them has a disseminated (stage 4) and another has a localized (stage 2) neuroblastoma. We observed subtelomeric locus D1Z2 (1p36) deletion in both tumors by using double-color fluorescence in situ hybridization. The MYNC gene was found in single copy in both tumors. Loss of heterozygosity (LOH) and restriction fragment length polymorphism analyses were performed by using DNA from frozen tumor cells and from microdissected tumor areas excised from paraffin-embedded sections. We detected somatic LOH at locus D1S468 (1p36) in a tumor-cell population with a trisomy 1 of the stage-2 patient. Neuroblastoma cells of the stage-4 patient were diploid and showed allelic loss at the following loci: D1S172, D1S80, D1S94, D1S243, D1S468, D1S214, D1S241, and D1S164. Haplotype study showed that the siblings inherited the same paternal 1p36--pter chromosome region by homologous recombination and that, in the two tumors, arm 1p of different chromosomes of maternal origin was damaged. Our results suggest that the siblings inherited the predisposition to neuroblastoma associated with paternal 1p36 region and that tumors developed as a consequence of somatic loss of the maternal 1p36 allele.

Background The human epidermal growth factor receptor 2 (HER2) and p53 pathways may be involved in chemotherapy sensitivity and/or resistance. We explore the value of HER2 and p53 status to foretell docetaxel sensitivity in advanced breast cancer. Methods HER2 and p53 expression was analysed in 36 (median age 55 yrs; range 37-87) metastatic breast cancer patients receiving docetaxel-based first-line chemotherapy. HER2 was determined by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), p53 was tested by IHC. We correlate the expression of study parameters with pathologic parameters, RECIST response and survival. The standard cut-off value of 2 was used to determine HER2 overexpression while p53 mean expression level was used to divide low/high expressors tumors. Results Median time to progression and overall survival were 9 (range 2 - 54) and 20 (range 3 - 101) months. Overall response rate was 41.6%. Nine cases showed HER2 overexpression. HER2 was more frequently overexpressed in less differentiated (p = 0.05) and higher stage (p = 0.003) disease. Mean FISH-HER2 values were significantly higher in responder than in non-responder pts (8.53 ± 10.21 vs 2.50 ± 4.12, p = 0.027). Moreover, HER2 overexpression correlates with treatment response at cross-tabulation analysis (p = 0.046). p53 expression was only associated with higher stage disease (p = 0.02) but lack of any significant association with HER status or docetaxel response. No significant relation with survival was observed for any parameter. Conclusion Our data seem to indicate that FISH-determined HER2 status but not p53 is associated with docetaxel sensitivity in metastatic breast cancer.

Xenon can induce cell and organ protection through different molecular mechanisms related to oxygen level. We explored the effect of xenon on oxygen-related signalling in the central nervous system via hypoxia inducible factor 1 alpha (HIF-1α) and mammalian target of rapamycin (mTOR). Methods. Postnatal day 7 (P7) Sprague Dawley rats were exposed to 25% oxygen/75% nitrogen (air group) or 25% oxygen/75% xenon (treatment group) for 120 min. Brains were collected immediately (transcript analysis—relative real-time polymerase chain reaction) or 24 hours (protein analysis—immunohistochemistry) after the 120-minute exposure period; peak anesthetic preconditioning has been previously identified at 24 hours post-exposure. Results. HIF-1α transcript and protein levels were found to be increased in xenon-exposed compared to air-exposed brains. Sustained nuclear translocation of the protein, accounting for an increased activity of HIF-1α, was also noted. mTOR transcript analysis revealed no significant difference between xenon-exposed and air-exposed brains immediately after the 120-minute exposure. Conclusion. Our data suggest that xenon induces the upregulation of HIF-1α transcription and translation, which may contribute to xenon's neuroprotective preconditioning effect. However, given that xenon exposure did not affect mTOR transcription, further investigation into other signalling cascades mediating xenon’s effects on HIF-1α in developing brain is warranted.

Ductal pancreatic carcinoma (DPC) is a deadly disease with an incidence of 9 cases in 100,000 people per year and a mortality rate close to 100%. Allelic losses in the long arm of chromosome 9 are commonly encountered in many human malignancies but no data are yet available about DPC. We screened 40 laser-microdissected DPC samples and 6 pre-invasive lesions for 9 microsatellite mapping markers of region 9q21.3 through 9q34.2. A small overlapping region of deletion, spanning 8 million base pairs, was identified between D9S127 and D9S105. Two genes, RSG3 and KLF4, mapped to 9q31.1 through 9q32, were further investigated. A highly significant association was found between KLF4 gene expression levels and genomic status. Similarly, absence of immunohistochemical expression of KLF4 protein was found in 86.8% cases of DPC (33/38). Overexpression of KLF4 in a human pancreatic carcinoma cell line induced a significant decrease in the proliferation associated with up-regulation of p21 and the down-regulation of cyclin D1. In conclusion, we identified a novel oncosuppressor region located at the 9q 31.1-3 locus that is lost in DPC at high frequency. Loss of KLF4 expression is closely related to the genomic loss, and its restoration inhibits cancer cell proliferation, suggesting a key suppressor role in pancreatic tumorigenesis.

The transcription factor Krüppel-like factor 4 (KLF4) may act both as an oncogene and a tumor suppressor in a tissue-dependent manner, and further studies on its role in pancreatic ductal adenocarcinoma (PDAC) progression and clinical outcome are warranted. Therefore, we investigated the loss of heterozygosity (LOH) in the 9q22.3-32 region and loss of KFL4 gene expression in epithelial cells from 35 PDAC, 6 pancreatic intraductal neoplasias (PanINs) and 6 normal ducts, isolated by laser microdissection, as well as their correlation with overall survival (OS) in patients treated with gemcitabine in the adjuvant setting. LOH was evaluated with 4 microsatellite markers and in situ hybridization, while KLF4 expression was studied by reverse transcription-PCR and immunohistochemistry. LOH in at least 1 locus was observed in 25 of 35 PDAC cases and in 5 of 6 PanINs, respectively. In particular, the loss of the D9S105 marker was present in 46.9% of PDAC and 83.3% of PanINs, becoming the most deleted marker, while no LOH in D9S105 was observed in normal Wirsung pancreatic duct. Lack of KLF4 mRNA expression was significantly associated with: (1) genomic deletion flanking KLF4 in PDAC and in PanINs (with LOH of D9S105), (2) low-grade PDAC-associated PanIN, (3) lack of KLF4 protein expression, and (4) shorter OS. These results strongly suggest a relationship between D9S105 deletion and downregulation of KLF4 gene expression as an early event in PDAC progression, as well as a possible role of KLF4 as a prognostic biomarker in gemcitabine-treated patients. and IAP.

A case of renin-secreting juxtaglomerular cell tumour which presented with a severe hypertensive crisis and did not respond to angiotensin converting enzyme inhibitors but was promptly controlled by intravenous labetalol is reported. The diagnostic difficulties which can be encountered in such cases and the usefulness of the different diagnostic tests, including renal vein renin measurement, are discussed.

This article reviews the conventional clinicopathological, as well as the principal new emerging prognostic factors of breast cancer and proposes a tumor marker utility grading system for their use. In spite of the many advances in molecular biology toward better defining the biological aggressiveness of the primary malignancy, the conventional node-negative status, tumor size and grade are still the strongest predictors of relapse-free survival and/or overall survival. Microvessel count and bone-marrow micrometastases, among the more recently studied clinicopathological prognostic factors, and amplification and/or p53 mutation and S-phase fraction among the biological ones must be considered investigational, although, with enough documentation recommending their usefulness. Estrogen and/or progesterone expression, c-erbB-2 amplification and/or mutation are the prognostic factors currently included in the principal clinical guidelines. They also enable probable forecast of the response to endocrine treatment or chemotherapy. In particular, c-erbB-2 is used to define the different risk categories of node-negative operated breast cancer patients. In recent years, microarray and quantitative reverse-transcription PCR technologies have enabled the study of multiple genetic alterations and computer algorithms have been developed for visual recognition of tumors that share so-called ‘signatures’. So far, different gene-expression patterns with different prognoses have been identified but methodological problems remain to be solved prior to routine use.

Objectives Pancreatic cancer still remains a challenge for its biological complexity and lack of effective therapeutic strategies. Establishing new pancreatic cancer cell lines is therefore of paramount importance to clarify its biology. Methods We established and characterized 4 new pancreatic cancer cell lines (PP78, PP109, PP117, and PP161) according to their genetic (K-Ras, TP53, CDKN2A, and MADH4; DNA fingerprinting; karyotype), cytostructural (cytokeratins 7, 8, 18, and 19 vimentin, and ezrin), and functional profiles (doubling time; migration assay). Results K-Ras, TP53, and CDKN2A gene alterations were detected in all 4 of them. Each cell line had a unique DNA profile revealed by DNA fingerprinting. A complex karyotype with numerous structural and numeric chromosomal abnormalities was present in each cell line. All 4 cell lines showed positivity for cytokeratins 7, 8, and 18. All but PP78 expressed cytokeratin 19, whereas vimentin was expressed only in PP117 and PP78 cells. A different ezrin cellular distribution was noticed in PP78 and PP117, being mostly located at membrane ruffles. This peculiar distribution was associated with the strongest migratory capability. Conclusions Our results seem to confirm the pancreatic ductal adenocarcinoma heterogeneity; in fact, the same genetic abnormalities (K-Ras, TP53, and CDKN2A) may have different effects on tumor biology depending on cellular differentiation.

We evaluated the possibility of prolonged chimerism formation in fetus and lamb, following human cord blood-selected CD133+ hemopoietic stem cell (HSC) transplantation into the celomic cavity of ewes at a pre-immune fetal age (44-45 days of pregnancy). Nineteen ewes were injected with HSC and 5 controls with a saline solution. By PCR, HLA-DQ alpha 1 and 6 human microsatellites (CODIS) were used for HSC traceability. FISH analysis was performed with 8 human DNA probes from different chromosomes, to confirm chromosomal integrity, nuclear DNA localization and donor DNA identification. Immunological staining for revealing HLA-DQ alpha 1 expression demonstrated multilineage engraftment. Both HLA-DQ alpha 1 and microsatellites were detected in different tissues of 3 available aborted fetuses, to a lesser extent in 11 lambs tested at 2-months, but not 12-months after birth. Although only 1 fetus of siblings of each sheep was injected, all siblings revealed positive engraftments. Microsatellite analysis showed evidence of human allele segregation in different tissues of individual fetuses and lambs. FISH analysis confirmed chimerism and the presence of human chromosomes. Non-detection of some human gene sequences in different chromosomes and random finding of allele segregation for some human heterozygous microsatellites were found in different tissues of individual animals. Controls born from un-transplanted ewes never revealed any human DNA sequences nor HLADQ alpha 1 expression.

Xenon and other inhalational agents induce cell and organ protection through different and only partially elucidated molecular mechanisms. Anesthesia induced or pharmacologic preconditioning is a recognized mechanism of cell protection. In this study we explored the gene transcription of activity-dependent neuroprotective protein (ADNP) in neonatal rat brain as consequence to xenon exposure, comparing the noble gas to nitrogen. Seven-day-old Sprague Dawley rats were exposed for 120 min to 75% xenon and 25% oxygen or control condition consisting of 75% nitrogen and 25% oxygen (Air). ADNP was found to be differentially expressed by SSH, validated by Relative Real-Time PCR (RT-PCR) and confirmed by western blot and immunohistochemistry. The differential expression of ADNP in the rat neonatal brain may account for the preconditioning and neuroprotective effects exerted by gas xenon.

The PI3K-Akt cascade is a key signaling pathway involved in cell proliferation, survival, and growth. Activating PIK3CA mutations have been reported in breast carcinoma (BC). The aim of this study was to characterize the PIK3CA mutations at exons 9 and 20 in a series of 176 sporadic and 22 hereditary BCs and to correlate the results with clinicopathologic parameters and survival. In sporadic BC, 68 missense mutations were detected. PIK3CA mutations were significantly associated with ER+ in HER2-negative cases. A higher frequency of PIK3CA mutations was present in lobular carcinoma compared with ductal carcinoma (50% vs. 35%). There was no association between the survival and PIK3CA mutational status. In hereditary BC, PIK3CA mutations were found only in the BRCA2 group. The PIK3CA mutation seems to characterize the luminal-type BC, in both sporadic and BRCA2 mutated forms, and is absent in the basal-type BC, in both the sporadic and BRCA1 mutated forms.

BACKGROUND. Neuroblastic tumors (NTs) are largely comprised of neuroblastic (Nb) cells with various quantities of Schwannian stromal (SS) cells. NTs show a variable genetic heterogeneity. NT gene expression profiles reported so far have not taken into account the cellular components. The authors reported the genome-wide expression analysis of whole tumors and microdissected Nb and SS cells. METHODS. The authors analyzed gene expression profiles of 10 stroma-poor NTs (NTs-SP) and 9 stroma-rich NTs (NTs-SR) by microarray technology. Nb and SS cells were isolated by laser microdissection from NTs-SP and NTs-SR and probed with microarrays. Gene expression data were analyzed by the Significance Analysis of Microarrays (SAM) and Game Theory (GT) methods, the latter applied for the first time to microarray data evaluation. RESULTS. SAM identified 84 genes differentially expressed between NTs-SP and NTs-SR, whereas 50 were found by GT. NTs-SP mainly express genes associated with cell replication, nervous system development, and antiapoptotic pathways, whereas NTs-SR express genes of cell-cell communication and apoptosis. Combining SAM and GT, the authors found 16 common genes driving the separation between NTs-SP and NTs-SR. Five genes overexpressed in NTs-SP encode for nuclear proteins (CENPF, EYA1, PBK, TOP2A, TFAP2B), whereas only 1 of 11 highly expressed genes in NTs-SR encodes for a nuclear receptor (NR4A2). CONCLUSIONS. The results showed that NT-SP and NT-SR gene signatures differ for a set of genes involved in distinct pathways, and the authors demonstrated a low intratumoral heterogeneity at the mRNA level in both NTs-SP and NTs-SR. The combination of SAM and GT methods may help to better identify gene expression profiling in NTs. Cancer 2008. © 2008 American Cancer Society.

OBJECTIVES The most frequent genomic abnormality in clear cell renal cell carcinoma (cc-RCC) is inactivation of Von Hippel-Lindau gene (VHL). pVHL19 is a ligase promoting proteosomal degradation of hypoxia-inducible factor-1alpha (HIF-1alpha); pVHL30 is associated with microtubules. VHL exert its oncogenetic action both directly and through HIF-1alpha activation. TNM classification is unable to define a correct prognostic evaluation of intracapsular cc-RCC. The nucleo-cytoplasmic trafficking in VHL/HIF-1alpha pathway could be relevant in understanding the molecular pathogenesis of renal carcinogenesis. This study analyzes VHL/HIF-1alpha proteins in a large series of intracapsular cc-RCCs, correlating their expression and cellular localization with prognosis. MATERIALS AND METHODS Two anti-pVHL (clones Ig32 and Ig33) and 1 anti-HIF-1alpha were used on tissue microarrays from 136 intracapsular cc-RCCs (mean follow-up: 74 mo). Clone 32 recognizes both pVHLs, whereas clone 33 only pVHL30. Results were matched with clinicopathologic variables and tumor-specific survival (TSS). RESULTS A strong cytoplasmic positivity was found for all antibodies in the largest part of cases, associated to a strong nuclear localization in the case of HIF-1alpha. All pVHL-negative cases were associated with high HIF-1alpha expression. pVHL negativity and HIF-1alpha nuclear positivity significantly correlated with shorter TSS. In multivariate analysis both pVHL negativity and HIF-1alpha nuclear expression were independent predictors of TSS. CONCLUSIONS The localization of the proteins well matches with their role and with the supposed tumor molecular pathways. The correlation with prognosis of VHL/HIF-1alpha alterations confirms the relevance of their molecular pathway and of the cellular trafficking of their products in the pathogenesis of renal cancer.

Fluorescent in situ hybridization (FISH) is a valuable method for determining Her-2/neu status in breast carcinoma samples, an important prognostic indicator. Visual evaluation of FISH images is a difficult task which involves manual counting of dots in multiple images, a procedure which is both time consuming and prone to human error. A number of algorithms have recently been developed dealing with (semi)-automated analysis of FISH images. These algorithms are quite promising but further improvement is required in improving their accuracy. Here, we present a novel method for analyzing FISH images based on the statistical properties of Radial Basis Functions. Our method was evaluated on a data set of 100 breast carcinoma cases provided by the Aristotle University of Thessaloniki and the University of Pisa, with promising results.

Background Human Papilloma Virus (HPV) is a DNA virus belonging to the Papovavirus family. Genital HPV types have been subdivided into medium-low risk, and high-risk (HPV 16 and 18), frequently associated with cervical cancer. Three DNA-based piezoelectric biosensors were here developed for a quick detection and genotyping of HPV. Methods We developed a method for the detection and genotyping of HPV in human cervical scraping samples based on coupling DNA piezoelectric sensors with Polymerase Chain Reaction (PCR). The novelty of this work was the design and immobilisation of a degenerate probe (chosen in a conserved region of the viral genome) for the simultaneous detection of 16 virus strains and of two specific probes (chosen in a less-conserved region of the viral genome) for genotyping. Results The three biosensors were optimised with synthetic oligonucleotides with good reproducibility (HPVdeg CV% av 9%, HPV16 CV%av 9%; HPV18 CV%av 11%) with a detection limit of 50 nM. Cervical scraping samples after PCR amplification (in 40–200 nM range), were tested without the need of label with high selectivity and reproducibility. The results were in agreement with a reference method used in routinary analysis. Conclusion Piezoelectric biosensors have proven to be suitable for detection and genotyping of HPV.

Protein markers are commonly used in forensic medicine to establish the origin of human fluids detected in crime scenes. Semenogelins, the major protein constituents of semen coagulum, are the most effective markers for semen detection. Recently, it has been demonstrated that semenogelins are also ectopically expressed in small cell lung carcinomas (SCLC) and in a minority of non-small cell lung carcinomas (NSCLC). This finding prompted us to look for semenogelin expression in the serum of lung cancer patients. A set of 13 serum samples (3 from SCLC and 10 from NSCLC patients) was screened by enzyme-linked immunosorbent assay (ELISA), using a commercially available kit. Four of the NSCLC cases showed positive results. Ectopic expression of marker proteins in individuals affected by cancer could represent a potential source of forensic pitfalls.

Protein markers are commonly used in forensic medicine to establish the origin of human fluids detected in crime scenes. Semenogelins, the major protein constituents of semen coagulum, are the most effective markers for semen detection. Recently, it has been demonstrated that semenogelins are also ectopically expressed in small cell lung carcinomas (SCLC) and in a minority of non-small cell lung carcinomas (NSCLC). This finding prompted us to look for semenogelin expression in the serum of lung cancer patients. A set of 13 serum samples (3 from SCLC and 10 from NSCLC patients) was screened by enzyme-linked immunosorbent assay (ELISA), using a commercially available kit. Four of the NSCLC cases showed positive results. Ectopic expression of marker proteins in individuals affected by cancer could represent a potential source of forensic pitfalls.

A case of incomplete VATER syndrome (esophageal atresia, anorectal malformation, vertebral anomalies, horseshoe kidney) associated with a foregut duplication of the esophagus is described. A review of the literature is presented.

A permanent cell line, BO-101, was derived from a classic vermian medulloblastoma in a 9-year-old child. This line grew in vitro in adherent cultures and grew in athymic mice as serially transplantable intracranial and subcutaneous xenografts. Intracranial neoplasms grew as masses of small cells, which focally showed large cells with intense immunoreactivity for desmin, myoglobin and α-striated actin. The rhabdomyoblastic nature of these cells was confirmed ultrastructurally. The primary neoplasm showed immunoreactivity for synaptophysin, neuron-specific enolase and vimentin. A large panel of monoclonal antibodies and antisera against neuronal and glial antigens failed to show glial and neuronal immunoreactivity in the cell culture and xenografts. Despite the marked genotypic and phenotypic differences, the original neoplasm and the cell line share a common chromosomal marker del (12) (p 13.1). The BO-101 line differs phenotypically and genotypically from previously established medulloblastoma cell lines and further supports the heterogeneous biologic proprieties of the cell populations that constitute these neoplasms.

1124 Background: HER2 overexpression predicts response to trastuzumab in early and advanced breast cancer. Preliminar evidences indicate that HER2 signaling pathway may also influence chemotherapy ...

ZusammenfassungZwischen Januar 1984 und Dezember 1988 wurden 107 Neuroblastom (Nb)-Frischtumoren (inklusive 53 Patienten weniger als 1 Jahre alt zur Zeit der Diagnose) auf N-myc Amplifikation (NMA) untersucht. Zusatzlich wurde NMA mit u. a. folgenden fur die Prognose wichtigen biologischen und klinischen Variablen korreliert: Klinisches Stadium [1,2], histologisches Grading [3], urinare Ausscheidung von Vanillinmandelsaure (VMA) und Homovanillinsaure (HVA); Serumspiegel von Ferritin (Fer), neuronspezifische Enolase (NSE) und Laktatdehydrogenasen (LHD); und Volumen (Vol) des primaren Tumors. Diese Korrelationen wurden unter allen 107 Patienten und in der Sauglingsgruppe (Alter ≤ 1 Jahr) ausgewertet.N-myc war amplifiziert in 7/53

Small-cell osteosarcoma is an entity which shares some clinical and pathological features with both classic osteosarcoma and Ewing's sarcoma of bone. While noted to be "not radiosensitive" when first described, a retrospective review the National Cancer Institute experience of five patients with small-cell osteosarcoma treated with radiation therapy following biopsy (three pts) or limited excision (two pts) showed local control in all five patients with two long-term disease-free survivors (12, 18 years). This compares to three patients treated with surgery alone where one patient failed locally and one patient is a long-term disease-free survivor (7 years). We have studied the in vitro radiation response of a recently established small-cell osteosarcoma cell line (TC-252) and compared its response with that of a classic osteosarcoma cell line (U2-OS) and an Ewing's sarcoma cell line (5838). The small-cell osteosarcoma line responded with a similar Do and extent of PLDR compared to the Ewing's line and was different from the in vitro radiation response of classic osteosarcoma. Based on this small clinical series and the in vitro radiation studies, we conclude that small-cell osteosarcoma is a radioresponsive tumor. Definitive radiation therapy or conservative surgery plus radiation therapy are effective alternative therapeutic options, compared to ablative surgery, for the local treatment of this uncommon bone tumor of children and young adults.