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A range of different techniques are available for predictive biomarker testing for non-small-cell lung cancer (NSCLC) clinical management. International guidelines suggest next-generation sequencing (NGS) as the preferred procedure, but other reverse transcriptase-polymerase chain reaction (RT-PCR)-based methods are rapidly evolving. In this study, we evaluated the reliability and accuracy of the IdyllaTM GeneFusion assay, a rapid and fully automated platform able to simultaneously detect ALK, ROS1, RET and NTRK1/2/3 and MET ex14 skipping mutations and compared its performance with routine reference methods. The cohort included thirty-seven NSCLCs plus two parotid gland carcinomas, previously characterized for the above alterations through either IHC, FISH, RT-PCR or NGS. In 36 of 39 cases, the Idylla GeneFusion assay and the reference methods were concordant (overall agreement: 92.3%). Tumor sections stored at room temperature for up to 60 days and 17 cases older than 2 years were successfully characterized. Our results suggest that the Idylla GeneFusion assay is a reliable tool to define gene fusion status and may be a valuable stand-alone diagnostic test when time efficiency is needed or NGS is not feasible.

AimsDiagnostic tumour samples are mandatory for morphologic and molecular diagnosis of non-small cell lung cancer (NSCLC) to establish the best therapeutic approach. In the presence of small tumour tissue sample, the pathologist needs to make responsible choices to achieve a correct diagnosis and save material for subsequent molecular evaluations. Nevertheless, in some instances, the diagnostic process can lead to tissue depletion. The automated Idylla epidermal growth factor receptor (EGFR) mutation test has been developed to rapidly process formalin-fixed paraffin-embedded (FFPE) pathologic material, without previous DNA extraction. This study aimed to test whether this platform is suitable for the reuse of H&E, immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) diagnostic slides.MethodsA training set of 19 FFPE tissues with known EGFR status was revaluated on H&E slides. Fourteen of them were also tested using IHC and FISH treated specimens. An additional series of 25 H&E, IHC or FISH slides of NSCLC cases tested for EGFR mutation at an external institution was blindly assessed as a validation cohort.ResultsCombining the two sets, 32 of 32 classical ex19dels and p.L858R were correctly identified. Three uncommon mutations (p.G719X, p.L861Q and ex20ins) were also detected. Four discrepancies were related to rare ex19del/ins not included in the Idylla list of detectable mutations. Two p.T790M variants were missed on one FFPE and two H&E slides but were detected using IHC and FISH sections from the same FFPE blocks.ConclusionsThe Idylla EGFR mutation test is highly reliable using differently treated tumour specimens and should be validated in larger studies.

AimsHeterogeneous implementation of molecular tests in current diagnostic algorithm at a European and international level is emerging as a major issue for efficient lung cancer molecular profiling.MethodsFrom May 2017 until October 2017, N=1612 patients referring to 13 Italian institutions were selected, at advanced stage non-small cell lung cancer (NSCLC), and prospectively evaluated. Principal endpoints were: the percentage of diagnoses performed on cytological and histological material, the proportion of requests for epidermal growth factor receptor (EGFR) mutational status, and resistance mutations detected on tissue and/or liquid biopsy samples after first-generation or second-generation tyrosine kinase inhibitors, the proportion of requests for anaplastic lymphoma kinase (ALK) gene rearrangements, ROS proto-oncogene 1 (ROS1) and Kirsten Rat Sarcoma (KRAS) determinations, the proportion of requests for programmed death-ligand1 (PD-L1) evaluation and, finally, the different assays used for the detection of EGFR mutations, ALK and ROS1 gene rearrangements and PD-L1 expression.ResultsOf 1325 patients finally included, only 50.8% requests were related to driver mutations with target agents already available in first-line at that preplanned time, while 49.2% were associated with PD-L1, ROS1, KRAS and others. Multiplex genomic assays (such as next-generation sequencing) were considered by all participating centres.ConclusionsTo the best of our knowledge, this is the first study in a ‘real-life daily practice’ involving both pathologists and oncologists evaluating routinely workflow and trends towards improvements in molecular requests. Collected data aim to describe the applied algorithms and evolution of molecular screening for stage IV NSCLC in clinical practice.

Diagnostic tumour samples are mandatory for morphologic and molecular diagnosis of non-small cell lung cancer (NSCLC) to establish the best therapeutic approach. In the presence of small tumour tissue sample, the pathologist needs to make responsible choices to achieve a correct diagnosis and save material for subsequent molecular evaluations. Nevertheless, in some instances, the diagnostic process can lead to tissue depletion. The automated Idylla epidermal growth factor receptor (A training set of 19 FFPE tissues with knownCombining the two sets, 32 of 32 classical ex19dels and p.L858R were correctly identified. Three uncommon mutations (p.G719X, p.L861Q and ex20ins) were also detected. Four discrepancies were related to rare ex19del/ins not included in the Idylla list of detectable mutations. Two p.T790M variants were missed on one FFPE and two HE slides but were detected using IHC and FISH sections from the same FFPE blocks.The Idylla

The discovery of tyrosine kinase inhibitors (TKI) has remarkably improved the clinical course of patients with non-small cell lung cancer driven by Epidermal Growth Factor Receptor (EGFR) mutations. However, virtually in all cases, the disease resurfaces in a TKI-resistant form that is mainly linked to an acquired EGFR-T790M mutation, a MET amplification, or small cell lung cancer (SCLC) transformation. Third-generation TKIs are able to block tumor growth through an irreversible binding to the T790M-mutated receptor. Such new treatments require the diagnostic analysis of new pathologic tissue or a liquid biopsy to detect the presence of the T790M mutation.Pre-TKI and post-TKI biopsies from 27 patients with an activating EGFR mutation were collected and analyzed for EGFR-T790M mutation, MET amplification, and SCLC transformation.The T790M mutation was found in 16 patients (59%) whereas MET gene amplification was found in 2 (10.5%) of 19 evaluated cases. The histologic transformation from adenocarcinoma (ADC) to SCLC was identified in 3 patients (11%). In one of them reversal from SCLC back to adenocarcinoma was observed. One patient had the T790M mutation concordantly detected in 2 synchronous lesions whereas another patient showed T790M positivity only in one of 2 specimens. In 4 patients longitudinal biopsies revealed T790M gains and losses not always according to biological expectations.Intrapatient molecular or histologic heterogeneity may be frequently found during routine treatment of non-small cell lung cancer patients. This biological aspect may have profound repercussions on subsequent therapeutic decisions, and therefore requires in-depth investigation.

The rapid and fully automated IdyllaThe study was divided into two parts. In the first part, we carried out IdyllaThe IdyllaOur data, although preliminary, show that the Idylla

We identified a group of melanocytic lesions with an architectural pattern very similar to that of a junctional nevus: cells mostly grouped in distinct nests, more or less of the same size and shape, and regularly distributed along the dermoepidermal junction. In contrast with these nevus-like features, these neoplasms display additional details which are incompatible with a diagnosis of junctional nevus. These include areas of lentiginous array with focal pagetoid spread of melanocytes above the junction; marked cytological atypia, such as nuclear enlargement, hyperchromasia, nuclear membrane thickening and with a mild degree of cellular pleomorphism. Moreover, these lesions mostly developed on severely sun-damaged skin of old patients. Using a four-probe fluorescence in situ hybridization (FISH) assay targeting RREB1, MYB, Cep6, and CCND1, we found that seven of the eight propositus cases showed chromosomal aberrations consistent with the standardized FISH diagnostic criteria for melanoma. Instead, the five junctional nevi that served as controls were negative in this test. These findings underline the utility of correlating clinical-pathological observations with FISH analysis for diagnosing correctly as melanoma these malignant neoplasms, which closely simulate a junctional nevus.

Study Type – Therapy (individual cohort) Level of Evidence 2b What's known on the subject? and What does the study add? Sorafenib is an oral receptor tyrosine kinase inhibitor that inhibits RAF serine/threonine kinases and receptor tyrosine kinases (vascular endothelial growth factor receptors 1, 2, 3 and platelet-derived growth factor-beta, Flt-3 and c-kit) that are implicated in tumourigenesis and tumour progression. Sorafenib is approved for the treatment of advanced renal cell cancer. B-RAF mutations (V600E and K601E) may be interesting negative predictive markers in metastatic kidney cancer patients treated with Sorafenib. Larger trials are needed to validate this hypothesis and should include B-RAF molecular analysis for better patient selection. OBJECTIVE • To assess both clinical and biological efficacy and toxicity of sorafenib in patients with metastatic renal cell carcinoma (mRCC) previously treated with an anti-angiogenic vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor. METHODS • Sorafenib is an orally active multikinase inhibitor approved for the treatment of mRCC. • Drug-focused translational research on tissues (i.e. B-RAF) and plasma (VEGFR-α, circulating endothelial cells, endothelial progenitor cells) was performed to define biological predictive and prognostic markers and their related kinetics. • Patients with mRCC pretreated with an anti-angiogenic treatment, an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0–2 and adequate organ function were eligible. • Patients received sorafenib 400 mg twice a day continuously in 4-week cycles. Patients with no progressive disease at 12 weeks continued to receive sorafenib at the standard dose, whereas progressing patients received an increased dose (600 mg twice a day) with early disease restaging after 4 weeks. Patients who progressed at 600 mg twice a day went off study. Efficacy (overall tumour control) was assessed by Response Evaluation Criteria in Solid Tumors. RESULTS • In all, 19 patients were entered. The baseline characteristics were as follows: ECOG PS 0–1 94.8%; median (range) age 62 (41–81) years; nephrectomy 100%; surgery for metastatic disease 26.4%; clear cell 79.1%; papillary cell 15.7%; sarcomatoid/high grade 5.2%; two or more metastatic sites 84%. • Overall, 11 patients (58%) had disease control at 6 months without significant correlation between response to prior therapy and hypertension. Progression-free survival (PFS) of 8.3 months was observed. • Of six patients for whom the dose was escalated due to early progression, three benefitted with PFS of >3 months. Three (15.7%) of 19 patients had a V600E mutation and one had a K601E mutation; PFS appeared to be substantially shorter in these patients compared with 15 patients with wild-type B-RAF (2.5 vs 9.1 month, P < 0.05). • The most common toxicity (National Cancer Institute Common Toxicity Criteria, NCIC 3.0, all patients) was grade ≥1 diarrhoea and grade 2–3 hand–foot syndrome in 11 patients. Grade 3 mucositis was observed in one patient. CONCLUSIONS • Sorafenib at doses of 400–600 mg twice a day continuously results in acceptable and well tolerated salvage treatment after VEGFR tyrosine kinase inhibitor failure. • In progressive patients, treatment with a higher dose could be a valid option and B-RAF mutations may be an interesting predictive marker to be studied in a larger randomized trial.

// Alessandra Ulivieri 1, 2 , Giuseppe Cardillo 3 , Liborio Manente 1 , Gregorino Paone 4 , Andrea Petricca Mancuso 5 , Leonardo Vigna 5 , Enrico Di Stasio 6 , Rita Gasbarra 1 , Salvatore Girlando 7 , Alvaro Leone 1 1 Anatomic Pathology Unit, San Camillo-Forlanini Hospitals, Rome, Italy 2 Laboratory of Biomedical research “Fondazione Niccolo Cusano per la Ricerca Medico-Scientifica” Niccolo Cusano University of Rome, Rome, Italy 3 Thoracic Surgery Unit, San Camillo-Forlanini Hospitals, Rome, Italy 4 Department of Respiratory Diseases, San Camillo-Forlanini Hospitals, Rome, Italy 5 Department of Medical Oncology, San Camillo-Forlanini Hospitals, Rome, Italy 6 Institute of Biochemistry and Clinical Biochemistry, Universita Cattolica del Sacro Cuore, Rome, Italy 7 Anatomic Pathology Unit, S. Chiara Hospital, Trento, Italy Correspondence to: Alvaro Leone, e-mail: aleone@scamilloforlanini.rm.it Keywords: Pathology, melanoma, pulmonary metastases, BRAF, NRAS, EGFR Received: May 15, 2015 Accepted: June 20, 2015 Published: July 04, 2015 ABSTRACT Background: Melanoma is highly curable in early stages but holds devastating consequences in advanced phases with a median survival of 6–10 months. Lungs are a common metastasis target, but despite this, limited data are available on the molecular status of pulmonary lesions. Materials and Methods: 25 patients with surgically resected melanoma lung metastases were screened for BRAF, NRAS, CKIT and EGFR alterations. The results were correlated with time to lung metastasis (TLM), relapse-free survival after metastasectomy (RFS) and overall survival (OS). Results: BRAF or NRAS were mutated in 52% and 20% of cases while CKIT was unaffected. Chromosome 7 polysomy was detected in 47% of cases with 17.5% showing EGFR amplification and concomitant BRAF mutation. NRAS mutated patients developed LM within 5 yrs from primary melanoma with larger lesions compared with BRAF (mean diameter 3.3 ± 2.2cm vs 1.9 ± 1.1cm, p = 0.2). NRAS was also associated with a shorter median RFS and OS after metastasectomy. Moreover, Cox regression analysis revealed that NRAS mutation was the only predictive factor of shorter survival from primary melanoma ( p = 0.039, OR = 5.5 (1.1–27.6)). Conclusions: Molecular characterization identifies advanced melanoma subgroups with distinct prognosis and therapeutic options. The presence of NRAS mutation was associated to a worse disease evolution.

PREVALEAT II (PREmature VAscular LEsions and Antiretroviral Therapy II) is an ongoing multicenter, longitudinal cohort study aimed to the evaluation of cardiovascular risk in advanced naive HIV-infected patients starting their first HAART. It includes all consecutive naive pts with

To assess both clinical and biological efficacy and toxicity of sorafenib in patients with metastatic renal cell carcinoma (mRCC) previously treated with an anti-angiogenic vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor.Sorafenib is an orally active multikinase inhibitor approved for the treatment of mRCC. Drug-focused translational research on tissues (i.e. B-RAF) and plasma (VEGFR-α, circulating endothelial cells, endothelial progenitor cells) was performed to define biological predictive and prognostic markers and their related kinetics. Patients with mRCC pretreated with an anti-angiogenic treatment, an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0-2 and adequate organ function were eligible. Patients received sorafenib 400 mg twice a day continuously in 4-week cycles. Patients with no progressive disease at 12 weeks continued to receive sorafenib at the standard dose, whereas progressing patients received an increased dose (600 mg twice a day) with early disease restaging after 4 weeks. Patients who progressed at 600 mg twice a day went off study. Efficacy (overall tumour control) was assessed by Response Evaluation Criteria in Solid Tumors.In all, 19 patients were entered. The baseline characteristics were as follows: ECOG PS 0-1 94.8%; median (range) age 62 (41-81) years; nephrectomy 100%; surgery for metastatic disease 26.4%; clear cell 79.1%; papillary cell 15.7%; sarcomatoid/high grade 5.2%; two or more metastatic sites 84%. Overall, 11 patients (58%) had disease control at 6 months without significant correlation between response to prior therapy and hypertension. Progression-free survival (PFS) of 8.3 months was observed. Of six patients for whom the dose was escalated due to early progression, three benefitted with PFS of3 months. Three (15.7%) of 19 patients had a V600E mutation and one had a K601E mutation; PFS appeared to be substantially shorter in these patients compared with 15 patients with wild-type B-RAF (2.5 vs 9.1 month, P0.05). The most common toxicity (National Cancer Institute Common Toxicity Criteria, NCIC 3.0, all patients) was grade ≥1 diarrhoea and grade 2-3 hand-foot syndrome in 11 patients. Grade 3 mucositis was observed in one patient.Sorafenib at doses of 400-600 mg twice a day continuously results in acceptable and well tolerated salvage treatment after VEGFR tyrosine kinase inhibitor failure. In progressive patients, treatment with a higher dose could be a valid option and B-RAF mutations may be an interesting predictive marker to be studied in a larger randomized trial.

1 Anatomic Pathology Unit, San Camillo-Forlanini Hospital, Rome, Italy 2 Department of Cardiovascular and Respiratory Sciences, San Camillo-Forlanini Hospital, Rome, Italy 3 Thoracic Surgery Unit, San Camillo-Forlanini Hospital, Rome, Italy Medical Oncology Unit, San Camillo-Forlanini Hospital, Rome, Italy 5 1st Oncological Pulmonary Unit, San Camillo-Forlanini Hospital, Rome, Italy 6 2nd Oncological Pulmonary Unit, San Camillo-Forlanini Hospital, Rome, Italy

Gastrointestinal histopathology represents approximately half of the current work-flow in our laboratory, and six to ten of the reports signed out daily concern patients studied for Crohn’s disease (CD). This chapter summarizes the role of the pathologist in the management of patients with CD, starting from a glossary of the terms used in the information exchange between the pathologist radiologist gastroenterologist, and surgeon. This multidisciplinary team approach is crucial for the correct management of CD patients. The gastrointestinal tract has limited patterns of tissue response to triggers of inflammation and the diagnosis therefore relies upon the various combinations of these patterns with the clinical picture. The study of endoscopic specimens is based not only on a histopathological scheme but also on the presentation of the patient, which is of equal importance. Due to the limits in the information that can be extracted by studying the morphology of inflammation, molecular biology is expected to play an increasingly important role in providing the clinician with insight into the disease process in a single CD patient and thus in allowing the appropriate treatment strategy to be tailored accordingly.

The six members of the human prothymosin alpha gene family have been cloned and sequenced. One gene (PTMA) contains introns and appears to be the source of all isolated prothymosin alpha cDNAs. The remaining five genes are processed pseudogenes. Four of them have consensus TATA elements upstream of sequences nearly identical to the transcriptional start region of the intron-containing gene. Those four genes also contain open reading frames coding for proteins closely related to prothymosin alpha. In two of the pseudogenes, PTMAP2 and 5, the encoded proteins differ from the product of the parental gene at only two and four locations, respectively. The fifth pseudogene (PTMAP1) encodes a different protein owing to an upstream translational initiation start site and multiple deletions and insertions. Because the potential for expression exists in this system, a search for pseudogenomic transcripts was undertaken using the polymerase chain reaction to amplify reverse transcripts of mRNAs from many human tissues and bulk DNA from several human cDNA libraries. Evidence for pseudogenomic transcripts was not obtained. Therefore, we conclude that the human prothymosin alpha gene family contains only one functional gene.

Reduced expression of the nm23 gene in certain rodent model systems and human breast tumors has been correlated with high tumor metastatic potential. To investigate the functional effects of nm23 expression, we have transfected a constitutive murine nm23 -1 expression construct into highly metastatic K-1735 TK murine melanoma cells. TK clones expressing the exogenous nm23 -1 construct exhibited a reduced incidence of primary tumor formation, significant reductions in tumor metastatic potential independent of tumor cell growth, and altered responses to the cytokine transforming growth factor , in soft agar colonization assays, compared with control-transfected TK clones. In contrast, nm23 -1-transfected TK clones exhibited no significant differences in intrinsic tumor cell growth, i.e., primary tumor size in vivo, anchorage-dependent growth rate in vitro, and anchorage-independent colony formation in soft agar in vitro. The data demonstrate a suppressive effect of nm23 on several aspects of the cancer process, including tumor metastasis.

A new transperineal ultrasound-guided prostate rebiopsy method is described. The method is performed using a fixed grid approach similar to the method that has been in use for some time for brachytherapy i n prostate cancer. The method adopts special cylindrical cutting needles to take cores of varying lengths and was found to be more time consuming (this is the main reason it is mostly used for rebiopsies). It nevertheless allowed more accurate ard systematic mapping of the prostate gland than the standard method of taking multiple biopsies by sector. Sending in each biopsy fragment for separate histological examination based on grid coordinates allowed the Anatomical Pathology laboratory to build up an ideal topographic reconstruction of the disease lesions detected. The approach offers the advantages of a repeatable biopsy plan that can be compared with previous biopsies, more accurate spatial localisation of diseased tissue and accurate information on distribution within the gland. The benefits are multifold and all extremely interesting both for the diagnosis and subsequent choice, evaluation and administration of therapeutic treatment--and also for the dynamic study of evolving cell changes and cell structure anomalies detectable in the prostate gland.

The aim of this study was to evaluate nm23 expression as detected in malignant cells of neoplastic ascites and to verify its relationship with the presence of tissue nm23 and p53 in primitive neoplasia. Using an immunocytochemical assay with a specific anti-nm23 polyclonal antibody, nm23 expression was evaluated in ascitic effusions of 45 patients with ovarian serous adenocarcinoma and ascites in normal and/or hyperplastic mesothelial cells from 37 women with various neoplasms (12 ovarian neoplasms) free of malignant cells as controls. nm23 and p53 tissue expression was also detected in 21 corresponding tumor samples, including 11 bilateral lesions. nm23 was expressed in 57% of malignant effusions compared to 43% in controls: the two groups were not correlated. nm23 in effusions agreed with tissue expression (p=0.02) but a direct correlation was not demonstrated. The incidence of nm23 was more frequent in stage III than in stage IV disease (p=0.08) and was associated with mutated p53 expression (p=0.01). Using the Wilcoxon test for unpaired data, a higher incidence (p=0.05) of p53-positive tumors in bilateral cancers was found while a higher expression of nm23 in effusions of patients with monolateral lesions was observed (p=0.08). The presence of p53 was correlated with that of nm23 in both cytologic (p=0.005) and histological samples (p=0.01). Our findings, together with the diversity in biological behaviour present in various tumors, suggest that nm23 is a family of genes with differing biological functions which act as tumor-specific inhibiting factors within a complex process also involving other genes. Due to the analogies and correlations between nm23 and p53, the role of nm23 as a potential predictive factor of response to chemotherapy and in DNA repair is emphasized.

Gem is a small GTP-binding protein within the Ras superfamily whose function has not been determined. We report here that ectopic Gem expression is sufficient to stimulate cell flattening and neurite extension in N1E-115 and SH-SY5Y neuroblastoma cells, suggesting a role for Gem in cytoskeletal rearrangement and/or morphological differentiation of neurons. Consistent with this potential function, in clinical samples of neuroblastoma, Gem protein was most highly expressed within cells which had differentiated to express ganglionic morphology. Gem was also observed in developing trigeminal nerve ganglia in 12.5 day mouse embryos, demonstrating that Gem expression is a property of normal ganglionic development. Although Gem expression is rare in epithelial and hematopoietic cancer cell lines, constitutive Gem levels were detected in several neuroblastoma cell lines and could be further induced as much as 10-fold following treatment with PMA or the acetylcholine muscarinic agonist, carbachol.

e19033 Background: About 25% of pts will eventually develop lung metastases from primary melanoma. Activating mutations in BRAF, NRAS, CKIT and EGFR genes are involved in melanoma progression. Trials with drugs targeting oncogenic BRAF in melanoma have shown that a V600E mutation may identify a subgroup of pts with a high response rate. Methods: We analysedtheprevalence of BRAF, NRAS and CKIT mutations by direct sequencing and of EGFR copy number alterations by FISH, in pts affected by melanoma lung metastases, and explored their correlation with lung metastasis-free survival (LMFS) and post surgical progression-free survival (psPFS). Results: Over 10 years, 33 cases of pts with melanoma lung metastases were diagnosed at our institution, of which 22 underwent surgery with curative intent. The mean age was 64 years, and 19 (57.5%) were male. 51% carried a BRAF V600E mutation while 18% had a NRAS codon 61 mutation. The two events were mutually exclusive. No alterations were observed in exon 11 of the CKIT gene. FISH analysis indicated an EGFR low amplification in 3 cases and chromosome 7 polysomy in 13 cases. The presence of a BRAF V600E mutation was associated with a longer LMFS (median 6.1 vs 4.4 years, p

The majority of breast cancer patients succumb to metastatic disease. We summarize published and recent research concerning the nm23 gene in breast cancer metastasis. In a murine developmental study, nm23 expression increased with the functional differentiation of the mammary gland in nulliparous and pregnant animals. In human breast cancer, five studies have now demonstrated a significant association between reduced nm23 expression, at the RNA or protein levels, and aggressive tumor behavior. Nm23-negative tumor cells have been observed in comedo ductal carcinoma in situ lesions in two independent studies, indicating that decreases in nm23 expression begin prior to actual histologically identifiable invasion. Transfection studies, in which human nm23-H1 cDNA was expressed in the metastatic human MDA-MB-435 breast carcinoma cell line, indicate that nm23-H1 suppresses in vivo metastatic potential by 50-90%. Finally, our data in melanoma and breast carcinoma transfection systems suggest that the biochemical mechanism of nm23 suppressive activity is likely not due to its nucleoside diphosphate kinase activity, association with GAP proteins, or secretion from cells.

Tumor metastasis remains the leading cause of death for cancer patients, excluding those with skin cancers. Metastasis is a complex process, requiring tumor cell invasion, motility, arrest and extravasation from the circulatory system, angiogenesis, and avoidance of host immune responses. A considerable list of proteases, motility factors, and adhesion molecules for cells and extracellular matrix components have been examined for their potential roles in the tumor metastatic process [reviewed in 1,2].

4128 Background: Several studies suggest that increased gene copy number of the Epidermal Growth Factor Receptor (EGFR) or presence of mutations of the K-ras oncogene are associated with cetuximab ...

A direct method for mapping introns has been devised. The technique makes use of a radioactive synthetic RNA transcript of the gene and a complementary, single-stranded DNA copy of mRNA-derived sequences. Upon hybridization of the cDNA to RNA and cleavage with ribonuclease H, only exonic RNA sequences are degraded. The surviving RNA fragments are the introns. Electrophoretic analysis in denaturing agarose gels reveals the number and size of the introns. The order of the introns is determined separately using unlabeled RNA transcripts; surviving RNA fragments are transferred to a solid support and the blot is probed sequentially with a nested set of genomic RNA transcripts of the opposite strand. Using the human prothymosin α gene as an example, four introns were identified which from 5′ to 3′ were 2.6, 0.47, 0.47, and 0.28 kb in size. From mapping and sequencing experiments the sizes are 2.6, 0.465, 0.459, and 0.295 kb, respectively. Similarly, the presence of two 300-bp insertions in a human prothymosin α pseudogene was established; the inserts were later identified as 295-bp Alu repetitive elements.

Fas ligand (FasL) exists in transmembrane and soluble forms and induces apoptosis on cross-linking with the Fas receptor. We evaluated the biological significance of FasL and Fas in 61 tumor tissues and 9 cell lines of the Ewing's sarcoma family of tumors (ESFT). FasL was present in 62.5% and Fas in 79.4% of primary ESFT. Metastatic tumors had higher expression of FasL (95%), suggesting association with a metastatic phenotype. FasL was detected in the cytoplasm and membrane of ESFT cells by immunofluorescence. Western blotting revealed transmembrane and soluble FasL in cytosolic extracts and soluble FasL in conditioned media. Both transmembrane and soluble FasL induced apoptosis of Fas-sensitive Jurkat cells in co-culture experiments with ESFT cells or their media. Treatment with phenanthroline and the synthetic metalloproteinase inhibitor BB-3103 reduced the levels of soluble FasL in the media, suggesting that in ESFT, FasL is processed by a metalloproteinase and released in the extracellular milieu. The released soluble FasL may serve to attack cells of the immune system and/or interfere with the binding of transmembrane FasL with Fas, and results in down-regulation of transmembrane FasL. Synthetic metalloproteinase inhibitors may modify the ratio of transmembrane to soluble FasL.

Background: Fas ligand (FasL) is a transmembrane protein that induces apoptosis (programmed cell death) in susceptible cells by interacting with its receptor, Fas. Transmembrane FasL is cleaved by a metalloproteinase enzyme into a soluble form that is released into the extracellular medium. Tumors of the Ewing's sarcoma family express functional transmembrane FasL and release soluble FasL. This cleavage is inhibited by a matrix metalloproteinase inhibitor (MMPI). We therefore hypothesized that MMPIs can lead to apoptosis of tumor cells by inducing accumulation of transmembrane FasL. Methods: Ewing's sarcoma and neuroblastoma cell lines were treated with two synthetic MMPIs (BB-3103 and A-151011) and examined for apoptosis and expression of FasL and Fas. Results : Although MMPIs increase levels of FasL and Fas proteins on the surface of all tumor cells studied, they induced apoptosis in Fas-sensitive but not in Fas-resistant cell lines; the induction of apoptosis was inhibited by a Fas-neutralizing antibody. The increase in protein expression was not associated with enhanced transcription. Treatment with an MMPI sensitized the Ewing's sarcoma cells to Fas-activating antibody and to doxorubicin-induced apoptosis. Conclusions: MMPIs cause accumulation of transmembrane FasL by inhibiting its cleavage, accumulation of Fas (probably secondarily to FasL cleavage inhibition), and decreased levels of soluble FasL. These effects lead to apoptosis in Fas-sensitive cell lines. The observed cooperative action of MMPIs and doxorubicin suggests a possible role of MMPIs in combination treatments with standard apoptosis-inducing chemotherapeutic agents.