Human papillomavirus (HPV) testing is included in the cervical cancer screening program in the triage of women with equivocal (ASC-US) or low-grade (LSIL) cytological lesions. These women have an increased risk for developing high grade dysplasia and cancer (CIN2+) compared to women with normal cytology. However, in order to avoid unnecessary follow-up, as well as overtreatment, a high positive predictive value (PPV) of the triage test is important.The HPV test PreTect HPV-Proofer, detecting E6/E7 mRNA from the HPV types 16, 18, 31, 33 and 45, is used as triage test together with repeat cytology. PPV data for HPV E6/E7 mRNA testing during the period from January 2006 up to June 2009 are reported. In total, 406 of 2099 women (19.3%) had a positive HPV test result. Of the women with a positive test result and with a histological diagnosis (n = 347), 243 women had histological high-grade dysplasia or cancer (CIN2+), giving a PPV of 70.0% . For HPV 16 or HPV 33 positive women above 40 years of age, the PPV was 83.7%  and 84.6%  respectively. The PPV of test positive women with HSIL cytology was 94.2% .When the result in triage is HPV mRNA positive, our data suggest direct treatment for women above 40 years of age or for women with a concurrent cytological HSIL diagnosis, contributing to better clinical safety for these women. In addition, by decreasing the time to treatment, thereby reducing the number of recalls, the patient management algorithm will be considerably improved, in turn reducing follow-up costs as well as unnecessary psychological stress among patients. Cervical cancer can be prevented by early detection and treatment of precancerous lesions. In Norway, a cervical cancer screening program was introduced in 1995, recommending all women between 25 and 69 years of age to have a cytological cell sample (Pap-smear) every three years. Since the introduction of the program, the coverage of women taking a Pap-smear has increased and consequently, the rate of cervical cancer is reduced As a result of the finding that human papillomavirus (HPV) is a necessary cause of cervical cancer, HPV testing has been included in cervical cancer screening in several countries. In Norway, HPV testing has been included in secondary screening since 2005 : If the In Norway, several HPV tests for use in secondary screening are available. One of these tests is the PreTect HPV-Proofer assay , detecting HPV E6/E7 mRNA from the five most prevalent types causing cervical cancer. The University Hospital of North Norway has five years of experience with the use of this test. The argument for choosing this test in secondary screening was the need for a clinically specific test to find the women truly needing follow-up and essentially requiring referral to treatment. The HPV E6/E7 mRNA test has been shown to have a higher clinical specificity and positive predictive value (PPV) than HPV DNA tests The PPV reflects the proportion of patients with a positive test result who are correctly diagnosed and gives information about the accuracy of the test. At our hospital we have repeatedly observed that women with a cytological normal, ASC-US or LSIL lesion at triage, and who are HPV mRNA positive, actually have an underlying high-grade lesion or cancer which is revealed at colposcopy and biopsy. In addition, re-evaluation of the cytological or histological slides after a positive HPV test often reveals more severe cell-changes than first diagnosed. In a previous paper The Regional Committee for Research Ethics has approved the study. Written consent from the patients for their information to be stored in the hospital database and used for research was not needed because the data were obtained and analyzed anonymously.In the routine diagnostic practice at the University Hospital of North Norway, the HPV E6/E7 mRNA test PreTect HPV-Proofer  is used in the triage of women with an ASC-US or LSIL diagnosis for the detection of cervical dysplasia and cancer. The Department of Clinical Pathology receives cervical smears from the population of Troms and Finnmark County in Northern Norway. Approximately 23 000 cervical smears are analyzed each year and from January 2006 to June 2009, smears from 54 326 women aged 25–69 years were analyzed. A total of 2 858 women (5.3%) were diagnosed with ASC-US or LSIL. For these women, repeat cytology and the HPV mRNA test were recommended after six months. Liquid based cytology and HPV mRNA results were received from 2 099 women (73.4% of the 2 858 women). The reason for not obtaining HPV mRNA results for all the cases is the wide use of conventional Pap-smear rather than liquid based cytology, which does not allow for HPV analysis.Cells were extracted from the ThinPrep® 2000  for cytological examination. The HPV mRNA test uses a liquid based sample from the same material as cytology. The mRNA testing was performed according to the manufacturer's instructions  and in accordance with national guidelines for HPV testing .Cytological and histological diagnoses were obtained from the diagnostic database (SymPathy) at the Department of Clinical Pathology, University Hospital of North Norway. Biopsies with histological high-grade dysplasia or cancer (CIN2+) were evaluated by experienced pathologists. In Norway, the current clinical threshold for treatment is CIN2+; after histologically confirmed CIN2+, national guidelines recommend treatment by conization of the cervix. Women with benign or CIN1 histology are advised to be followed up with a new Pap-smear and HPV test after 6–12 months. Biopsies with uncertain cellular changes are immunostained with p16 in order to detect occult CIN lesions.It is important to note that in clinical practice, the guidelines are not consistently followed. In contrast to a well planned study, individual evaluations are frequently made and essentially the history of the patient is important in the assessment of whether or not she should be referred to treatment. Different aspects of deviations are included in the Numbers for PPV calculations are presented in For women being treated (n = 230), the diagnosis used for PPV calculations is the most high-grade histological diagnosis of the biopsy before treatment or of the cone after treatment; the biopsy before treatment may have therapeutic effect and therefore the diagnosis of the biopsy may be the correct one. The diagnosis of the biopsy is reported for 42 women; for 181 women, the diagnosis of the cone is reported. Seven women were referred to treatment directly after abnormal cytology and/or colposcopy and/or a persistent HPV result and for these women the diagnosis of the cone is reported.Of the total women with a positive HPV result and a histological diagnosis (n = 347), 243 women had histological CIN2+, giving a PPV of 70.0% . Of the The added value of the HPV mRNA test in detecting women with high-grade lesions is shown in In total, 13 women were found to have cancer , all of The main cause of invasive cervical cancer is the deregulated and persistent production of HPV E6 and E7 oncoproteins Our data show that when including all age groups, the HPV mRNA test has a PPV for CIN2+ of 70%. A previous data analysis The PPV of the diagnoses ASC-H and HSIL for CIN2+ are, according to Norwegian Cancer Registry data, 56.4% and 73.9% respectively Women with ASC-US or LSIL at repeat cytology and with a positive HPV mRNA test are referred directly to colposcopy and biopsy. When an HPV test is not used, women with ASC-US and LSIL are referred to colposcopy and biopsy after three times repetition of the diagnosis. This means that for women with an underlying high-grade lesion, the disclosure of their disease will be delayed with up to two years, which may be critical. The same applies to women with normal cytology at triage, for whom the PPV of HPV mRNA test positives for CIN2+ is 65.0%. Women with normal cytology (when the HPV test is not included) are referred back to the screening program, emphasizing the importance of an HPV test for these women. Of note is that the PPV for mRNA positive women with normal cytology is higher than the PPV for women with ASC-US . This maAn HPV mRNA positive HPV 16 result has a PPV for CIN2+ of 80.4% when including all age groups, representing a higher PPV than the cytological HSIL diagnosis. For the other HPV types, the PPV is somewhat lower. One explanation for the lower PPV of HPV 18 and HPV 45 may be the lower degree of noticeable cell changes related to these infections and the fact that lesions caused by these HPV-types often are located further up in the cervical canal (glandular lesions) The motivation for looking at the age group >40 years is related to the lower probability of children planned among women in this group. Given the impact of cervical treatment (such as LEEP conization) on pregnancy outcomes An added value of the HPV mRNA test in detecting women with high-grade lesions is presented in In total, 13 women  were diagnosed with cancer. Interestingly, the HPV test had an important role in detecting most of these women, explained by the knowledge that the cytological diagnosis is influenced by the HPV result and by the follow-up of women with normal or low-grade cytology due to a positive HPV result . ConsequTo conclude, our data suggest a clinical “test and treat” application of the HPV mRNA test: Direct treatment may contribute to a better clinical safety for test positive women above 40 years of age and for women with a concurrent cytological HSIL diagnosis. Consequently, by decreasing the time between testing and treatment, thereby reducing the number of recalls, the patient management algorithm will be considerably improved and will in turn reduce follow-up costs and more importantly, unnecessary psychological stress among patients will be avoided.


Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal.  These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning.  Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3.  Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation).  In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression).  The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.The nervous system of the marine mollusk Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5.  Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture.  The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.An additional advantage of the Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile  Prepare culture dishes. Coat glass well of Mattek glass-bottom culture dish with poly-l-lysine (made in sodium borate). Add enough to completely cover the glass well and leave for > 1hr (can be left overnight). Thoroughly remove the poly-l-lysine by rinsing in artificial seawater (ASW) 4-5 times. After removing the last rinse, add 2 mls of 50% L15 /50% hemolymph. This culture medium must be in dish for at least one hour prior to plating cells so that the hemolymph coats the dish. 20 and then place them for >1 hr under a UV light in the culture hood. Clean the tools and Sylgard dishes with ethanol, rinse them thoroughly with ddHUsing a microelectrode puller (e.g. Sutter Flaming Brown P-97), pull glass pipette electrodes with 1.5 mm outer diameter, 0.86 to 1.12 mm inner diameter and 100 mm length . Use parameters that produce an electrode with a long and wispy fine tip of such high resistance that there is no capillary intake of fluid when placed in solution. The presence of a fluid meniscus means that the electrode tip will damage neuron during isolation. The Sutter electrode cookbook provides excellent guidelines for setting up electrode programs. We use a box filament, and a one-step pull to generate culture electrodes. Prepare sharp electrodes for microdissection of neurons.Prepare anesthetic solution (0.35M MgCl2), culture medium , and protease solution . Aplysia for isolation of sensory neurons and of LFS motor neurons. Use juvenile 1-4 g Aplysia for isolation of L7 motor neurons. Remove the animals gently from the seawater tanks, and maintain in seawater  for no more than 30 minutes before beginning anesthesia and culture procedure.Animals: use adult 80-100 g 2 into adult Aplysia (80-100 g) using a 60 ml syringe with an 18 gauge 1.5 inch needle. Enter the foot of the animal at an angle of about 35 degrees, and do not enter too deeply. The goal is to inject into the hemocoel of the animal without penetrating the internal organs. The animal should become very distended and relaxed.Anesthetize the animals for removal of ganglia: Inject 0.35 M MgClDissect the ganglia: Pin the anesthetized animal on a dissecting dish, with a pin  at the head and the tail, and the foot facing up. Hold the foot with a toothed forceps and cut through the skin and underlying connective tissue the full length of the foot (from head to tail) with a surgical scissors. Pin the two sides down. Using a forceps and scissors, cut the esophagus and pull to the side to expose the ganglia. Using a fine forceps and fine scissors, cut out the pleural pedal ganglia . Leave a fair amount of nerve since this makes it easier to pin down the ganglia after protease treatment.Protease digestion of ganglia: Place the ganglia in protease solution (10 mg/ml of 1 unit/mg in L15) in an incubator set at 34.5°C (34-35°C is okay) for 2 hrs and 15min. Using the fine tip forceps to transfer and wash the ganglia in ASW three times. Keep the ganglia in L15. Note that the protease incubation time can vary. The purpose is to allow the connective tissue to be easily removed. If it is too difficult to desheath the ganglia without damaging the neurons, increase the protease incubation time. If it is extremely easy to desheath the ganglia, and the neurons are “soft,” reduce the protease incubation time.Desheathing the ganglia: Transfer the protease-digested ganglia to a 60 mm or 35mm dish containing sylgard and L15. Viewing under a stereo-microscope , remove pedal ganglia and pin down the pleural ganglia in the Sylgard dish in the proper orientation using insect pins and a fine forceps. With the fine forceps and a Vanna surgical scissors, carefully lift the connective tissue and cut it to expose the sensory cluster. Be careful not to ever touch or damage the neuronal somata. Remove any excess connective tissue from the dish and add more medium, making sure to never expose the desheathed ganglion to air.  The sensory cluster consists of approximately 200 clustered neurons of 40-50 micron diameter. Set a pin to make a “post” at a short distance from the ganglion (within the field of view).Isolation of neurons: Using long, sharp glass culture electrode, pull out the identified neurons one by one by touching the cell body just off center  and slowly and steadily pulling the cell soma, together with the axon, away from the ganglion. Gently tap against the pin to dislodge the neurons from the electrode. Plating neurons: Use a pipetman (P10) to transfer individual neurons from the Sylgard dish to a culture dish. Before transferring the neurons, pipet culture medium into the tip so that the plastic tip is coated with hemolymph (this prevents the neurons from sticking to the plastic). Take great care to avoid exposing the neurons to any air bubbles or to any extreme forces (i.e. very gently take up and remove the neurons from the pipetteman). Distribute the neurons evenly throughout the dish. Use a sharp electrode to gently straight the processes and tap them down to the bottom. Incubation: Leave the dishes on microscope stage at room temperature for no less than 3 hours. We routinely leave them overnight. Make sure that the microscope stage is unperturbed during this period. Cover the stage with aluminum foil to protect from light. Gently transfer them to an 18°C incubator. Cultures should adhere to the dish within approximately 3 hrs and new neurite growth should be visible within approximately 6-12 hrs. The growth of isolated sensory neurons reaches a plateau by DIV 3 or 4. Note that isolated sensory neurons doe not form autapses or chemical synapses with one another, although they do form electrical gap junctions.Aplysia, and there are approximately 20 LFS motor neurons per abdominal ganglion. The L7 motor neuron is isolated from juvenile (1-4 g) animals, and there is only one L7 per abdominal ganglia. LFS motor neurons are 40-50 microns in diameter, are interspersed among the LE sensory neurons close to the root of siphon nerve on the ventral surface of the abdominal ganglia, and are characterized by a subtle dark pigment spot in each soma. The L7 motor neuron is 100-150 microns in diameter and is present on the dorsal surface of the ganglion, on the middle edge of the left side of the ganglion. While it is more economical to use LFS motor neurons, the large size of the L7 motor neuron is advantageous for some experiments. The L11 motor neuron, also on the dorsal left surface of the abdominal ganglion, caudal to L7, is a nontarget motor neuron and can be effectively used as a control with which the sensory neuron fasciculates but does not form chemical synapses.Sensory neurons form synapse with target motor neurons in culture. The most commonly used motor neurons are the LFS motor neurons and the L7 motor neuron, from the abdominal ganglion. The LFS motor neurons are isolated from adult (80-100 g) 2 into juvenile Aplysia (1-4 g) using a 10 ml syringe with a 21-gauge needle.Anesthetize the animals for removal of ganglia. For LFS motor neurons, do as described for pleural sensory neurons. For L7 motor neurons, inject 0.35 M MgClDissect the abdominal ganglia: Pin the anesthetized animal on a dissecting dish as described above . Using a fine forceps and fine scissors, cut out the abdominal ganglia. Protease digestion of ganglia: This is done as described for pleural sensory neurons, except that the digestion time is reduced to 1 hr 45 min for ganglia from juvenile (1-4 g) animals. Desheathing the ganglia: Transfer the protease-digested ganglia to a 35 or 60 mm dish containing sylgard and L15. Viewing under a stereo-microscope , in the ganglia down in the Sylgard dish in the proper orientation using insect pins and a fine forceps. With the fine forceps and a Vanna surgical scissors, carefully lift the connective tissue and cut it to expose the neuronal cell bodies. To isolate the LFS motor neurons, pin the ganglion so that the ventral surface is facing up, and with a forceps pull the connective tissue sheath back to expose the entire half of the ganglion containing the LFS motor neurons (to your right), desheath the remaining parts of the ganglion, remove any connective tissue from the dish and add more L15. To isolate the L7 or L11 motor neuron, pin the ganglion so that the dorsal surface is facing up, and with a forceps pull the connective tissue sheath back to expose the half of the ganglion containing both L7 and L11 (to your left). Be careful not to ever touch or damage the neuronal somata. Place a pin to make a “post” at a short distance from the ganglion (within the field of view).Isolation of neurons: Remove neurons with a sharp electrode as described for the sensory neurons. The LFS neurons are differentiated from neighboring LE sensory neurons on the basis of a small dark pigment spot in their somata. The L7 neuron can be differentiated from the L11 neuron first because the L11 is caudal to L7, because the L11 cell body is more oblong in shape while the L7 cell body is rounder and because the L11 axon tends to branch into two close to the cell body. Plating neurons: Use a pipetman (P10) to transfer individual neurons from the Sylgard dish to a culture dish as described for the sensory neurons.Pairing with sensory neurons: Let the motor neurons adhere to the culture dish for at least 1 hr. Then add the isolated sensory neurons with a pipetman, delivering each sensory neuron adjacent to a plated motor neuron. Using a sharp glass electrode carefully straighten out the sensory neuron axon, and coax the sensory cell body to a position next to the motor neuron, at a distance equal to or slightly less than the length of the sensory cell axon. Using the glass electrode, coax the sensory axon to come into contact with the motor neuron, very gently using the side of the tapered electrode to finally have the sensory axon physically contact the axon of the motor neuron. Do not lift the culture dish, but rather gently glide the dish along the microscope stage.Leave the dishes on microscope stage at room temperature for no less than 3 hrs and transfer to an 18°C incubator as described for the sensory neurons.Cultures should adhere to the dish within approximately 3 hrs and new neurite growth should be visible within approximately 6-12 hrs. Sensory neurons form glutamatergic synapses with motor neurons very quickly--as soon as the cell are adherent enough to perform sharp electrode recording (3-5 hrs), an excitatory post-synaptic potential can be recorded. Synaptic connectivity continues to increase until DIV 3, and is then stable until DIV7. The neurons should show elaborate neurite outgrowth during the first one to three days in culture.Aplysia culture . It is collected from large (500 g-1 kg) animals, and the best time to collect hemolymph  is during the spring (mid-March to June). Swaddle the animal in a disposable underpad so that only a small portion of the animal is exposed, clean that portion of the skin with ethanol, and then hold it while another person uses a sterile razor blade to make an incision in the exposed area. Squeeze the animal so that the hemolymph squirts into a clean beaker, making sure that it does not contact the dirty skin of the animal. The hemolymph fills the hemocoel of the animal . Rewrap the animal once or twice, make a new incision and squeeze hard to collect as much hemolymph as possible . Hemolymph from each animal should be kept separately . Spin the hemolymph at 2000 x g for 10 min to remove blood cells. Aliquot the supernatant in 10 ml aliquots, label by animal and store at -80°C. Note that hemolymph stored at -20°C forms a precipitate in medium. A new aliquot of hemolymph must be used every time culture medium is prepared, and the aliquot should be thawed just prior to preparing the medium. Do not refreeze after thawing.Hemolymph is used as a growth factor in Aplysia sensory-motor cultures has a somewhat slow learning curve, since it involves the development of fine motor skills associated with microdissection and manipulation of individual neurons viewed through a stereo-microscope. In our experience, it takes approximately 1-3 weeks of practice to obtain healthy isolated sensory neurons in culture and an additional 1-3 weeks to learn how to pair sensory neurons with motor neurons. We routinely prepare cultures in a Labconco Clean Bench, although it is possible to prepare them on any laboratory bench or desk, as long as the microscope is unperturbed during the culturing . Since the neurons grow in seawater at 18°C, bacterial and fungal contaminations are significantly less common than in mammalian cell culture. The single most important variable in culture preparation is the quality of the animal. Aplysia can either be purchased from vendors who collect them directly from the Pacific Ocean , or from the University of Miami Mariculture facility, which collects breeding pairs from the Pacific and then grows Aplysia through one breeding cycle. As such, the animals are genetically heterogeneous, and, in the case of the animals collected from the ocean, they are also heterogenous in terms of their environmental history. There is some seasonality to the quality of neurons obtained from Aplysia, with the worst period usually occurring in August, and another lower quality period around December. Another important variable is the hemolymph. It is wise to test different batches of hemolymph for their growth-promoting capacity, and to use the same batch of hemolymph for a set of experiments.The successful preparation of Aplysia neuronal cultures, they possess a number of unique and valuable advantages to the study of synapse formation and synaptic plasticity. They form monosynaptic connections in culture that can be monitored by sharp electrode recording for periods of days. Well characterized protocols exist that elicit forms of plasticity that have clear parallels in the behavior of the animal. Stimuli can be applied to the bath or to subsets of synapses6,7, and the geometry of cultures can be varied so that one sensory neuron contacts a single motor neuron, or one sensory neuron with a bifurcated axon contacts two motor neurons, or two sensory neurons contact an individual motor neuron. Molecular and cell biological pathways can be altered in individual neurons by microinjection of DNA, RNA, siRNA and other reagents, and the effects on neuronal structure and function, synaptic transmission and plasticity can be monitored with temporal and spatial resolution. The NIH and Broad Institute are nearing completion of the sequencing of the Aplysia genome, which shouldDespite the difficulty of preparing


Background and purpose Periprosthetic bone loss after uncemented femoral hip revision is a matter of concern. We have used a proximally porous- and hydroxyapatite-coated prosthesis (Bi-Metric) in revision since 1989 and now we report the bone changes. This prosthesis is intended to distribute the forces more evenly and to avoid proximal femoral unloading.Methods 22 patients were unilaterally reoperated because of aseptic loosening. Only patients with a healthy contralateral hip were included. Mean age at revision was 69 (55–80) years. Bone defects were graded by Gustilo-Pasternak and Endo-Klinik classifications. Clinical assessment was performed with Harris hip score. We used radiographs and dual-energy X-ray absorptiometry to evaluate migration, femoral remodeling, and bone mineral density after 72 (30–158) months.Results The mean Harris hip score was 74 (30–100) points at follow-up. Mild thigh discomfort was present in 1 patient and moderate thigh pain in 3 patients. There was no loosening or subsidence. Osteolysis seen at revision had diminished in 19 of the 22 hips at follow-up. We noted a large reduction in bone mineral density. It was most pronounced in Gruen regions 1, 2, 6, and 7.Interpretation Revision with this stem is a reliable procedure; however, we noted a large degree of proximal bone loss that could lead to later mechanical complications or fractures. Uncemented hip revision after aseptic loosening has been promising in the short to medium time interval  and seveAll 60 patients who were reoperated at our department with the proximally porous and HA-coated tapered Bi-Metric stem between 1989 and 2002 due to aseptic femoral loosening were reviewed retrospectively. At follow-up 8 patients had died, 7 of whom had still had their stem in place. Only patients with a healthy contralateral hip were included (28 patients). 6 patients who got their index prosthesis because of a fracture (4 hips), inflammatory arthritis (1 hip), or dysplasia (1 hip) were excluded due to their different bone metabolism. 1 patient only attended the radiographic part of the study. This left 22 patients (22 hips) for the study.Mean age at revision was 69 (55–80) years and the mean follow-up time was 6 (2.5–13) years. The index diagnosis was primary osteoarthritis in all patients. 16 patients had had only 1 hip arthroplasty in the same hip before the revision, 4 patients had had 2 previous arthroplasties, and 2 patients had undergone 3 earlier arthroplasties. Mean time between the primary hip arthroplasty and our revision was 6 (1–13) years. The patients were reoperated with an uncemented prosthesis: the Bi-Metric femoral stem  . It is aThe patients were evaluated clinically, with interview and examination performed by one author (MS) who had not been involved in the operations. He categorized the patients' functional status according to Charnley's classification (1972). 13 patients had unilateral disease (group A), no patient had bilateral disease (group B) by virtue of selection criterion, and 9 patients were classified as group C, i.e. they had disabilities other than the hip, which interfered with their functional or locomotor capacity. Clinical outcome was assessed with Harris hip score (HHS). The patients were also asked if they suffered or had suffered mid-thigh pain. The pain was categorized as being mild, moderate, or severe.Standardized anterioposterior and lateral radiographs after the index operation were compared with radiographs before and immediately after the revision and with radiographs taken at the time of survey. All radiographs were reviewed by MS. He recorded bone deficiencies prior to revision, stem migration, bone remodeling, and periprosthetic femoral bone loss. Bone defects prior to revision were classified according to the The stability of the femoral component was assessed by the criteria of 2. The median change in BMD was expressed as a percentage of that on the unoperated side for the 7 regions.We measured the bone mineral density (BMD) of the periprosthetic femur in the coronal plane with a dual-energy X-ray absorptiometer, DEXA . During scanning, the patient was placed supine with standard knee and foot supports, and with the femur in neutral rotation. The scanner was equipped with software for femoral periprosthetic bone mineral measurement. The operated hip was scanned and BMD values in 7 Gruen regions of interest were analyzed. A simulated prosthesis mask was automatically superimposed on the unoperated side and adjusted, with the lesser trochanter as reference, to ensure that the same regions were scanned on both hips. The values were expressed as areal BMD, in g/cmInformed consent was obtained from each patient in the study. According to the ethics board at Karolinska Institute, no permission was required for this study (04-453/3). The investigation was approved by the coSORTtee for protection against radiation at Danderyd Hospital (2003-3).To estimate the precision error of the DEXA method, we had earlier made double measurements in 10 patients, with complete repositioning of the patient and the scanner. We found a precision error of 1–4% at the different Gruen regions .The Mann-Whitney U test for non-parametric variables (independent groups) was used to compare Charnley's functional class (A and C) with HHS, and to compare bone defects prior to revision with subsequent bone loss. The Wilcoxon signed-rank test (paired observations) was used to compare side differences in BMD. The statistical analyses were performed with the statistical package JMP . Differences were considered statistically significant at p-values < 0.05.The mean HHS for all patients was 74 (30–100) points. We noted a lower HHS among patients in Charnley's function class C (mean 58 points) compared to patients in class A (mean 86 points) (p = 0.003). 3 patients had moderate thigh pain and 1 patient had mild pain. 9 patients limped, 10 used walking support but 3 of these patients used a cane for long walks only.We saw no stem loosening after the rearthroplasty. Linear osteolysis at revision was evenly distributed in all regions and regression of osteolysis was more commonly noted in the distal regions . Signs oWe found a marked reduction in BMD on the reoperated femur in all regions compared to the unoperated side  ; the lar6 hips had dislocated, 3 of them more than once. 1 recurrent dislocation was successfully treated with exchange of liner and a larger head size. Another recurrent dislocation was treated with an orthosis for a few weeks after the last closed reduction and no dislocations occurred thereafter. The sixth patient with an unstable hip (recurrent) had cerebral palsy and has undergone 6 closed reductions with no dislocations in recent years. 1 trochanteric fracture occurred 6 months after revision; the fracture was diagnosed late and it was treated conservatively with a good result. 1 patient had a postoperative wound infection and 1 patient got a clinically apparent deep vein thrombosis. No case of pulmonary embolus, neurological or vascular complications was seen.The results after cemented revision arthroplasty are not encouraging . HoweverIn this paper, we report the bone loss around 22 implants after rearthroplasty with an uncemented hydroxyapatite-coated prosthesis due to aseptic loosening at a mean of 6 years. We have used this prosthesis in cases where the bone stock could give direct rigid stability for the implant, i.e. in revisions where the bone deficiencies were mild to moderate. Clinically, the patients in this study did well after the revision, achieving a mean HHS of 74 points at follow-up.In our series, only 2 femoral components subsided but they also showed several radiographic signs of stable fixation. We saw few reactive lines and more than half of the femurs showed formation of endosteal bone bridging (spot welds); these are considered to be associated with good fixation and osseointegration of uncemented stems .Stress-shielding is a well-known phenomenon around uncemented stems. The Bi-Metric stem is designed to reduce the stress-shielding effect by a porous coating aimed at osseointegration only in the proximal region. However, our radiographic data revealed that this is not the case. We saw several signs of stress-shielding, such as calcar resorption (8 hips) and distal cortical hypertrophy (6 hips). Distal cortical hypertrophy and proximal resorption are consequences of stress transfer; the frequencies are in accordance with previously reported remodeling around the same stem in primary arthroplasty at 5 years postoperatively . Linear Bone mineral measurement after an uncemented re-arthroplasty is an important means of investigating the reaction upon the bone after implantation of the stem. Of particular interest is the determination of whether new bone is forming or whether pre-existing bone is being resorbed in response to the altered patterns of stress that are created by the components. The use of an uncemented prosthesis in revision surgery also presents the opportunity for restoration of lost bone stock with grafting . Bone waA large loss of BMD in the proximal femur has been reported by many authors in longitudinal studies following primary uncemented arthroplasty . In the The use of an uncemented prosthesis in revision surgery presents the opportunity for restoration of lost bone stock with grafting . In anotThere are some limitations to our investigation. It is a cross-sectional study, where periprosthetic BMD is compared with that on the healthy side. This could be a cause of error, because of a possible side difference in BMD before revision. Thus, the BMD difference found after rearthroplasty in cross-sectional studies may be overestimated and only a longitudinal study would give more accurate information about the remodeling process. In retrospective studies, pre- and peroperative BMD have been ignored because preoperative BMD of the involved hip is not available for comparison. Reduced activity of the affected leg, both pre- and postoperatively, can contribute to differences —as can tIn summary, we found good medium-term clinical and radiographic results with the uncemented Bi-Metric femoral stem prosthesis in revision. Compared to primary THA, a similar remodeling pattern was seen but a larger and more widespread degree of bone loss in the proximal femur was noted with DEXA after rearthroplasty. Long-term results will show whether this could lead to mechanical complications such as loosening or fractures.


Misfolded proteins are generally recognised by cellular quality control machinery, which typically results in their ubiquitination and degradation. For soluble cytoplasmic proteins, degradation is mediated by the proteasome. Membrane proteins that fail to fold correctly are subject to ER associated degradation (ERAD), which involves their extraction from the membrane and subsequent proteasome-dependent destruction. Proteins with abnormal transmembrane domains can also be recognised in the Golgi or endosomal system and targeted for destruction in the vacuole/lysosome. It is much less clear what happens to membrane proteins that reach their destination, such as the cell surface, and then suffer damage.We have tested the ability of yeast cells to degrade membrane proteins to which temperature-sensitive cytoplasmic alleles of the Ura3 protein or of phage lambda repressor have been fused. In soluble form, these proteins are rapidly degraded upon temperature shift, in part due to the action of the Doa10 and San1 ubiquitin ligases and the proteasome. When tethered to the ER protein Use1, they are also degraded. However, when tethered to a plasma membrane protein such as Sso1 they escape degradation, either in the vacuole or by the proteasome.Membrane proteins with a misfolded cytoplasmic domain appear not to be efficiently recognised and degraded once they have escaped the ER, even though their defective domains are exposed to the cytoplasm and potentially to cytoplasmic quality controls. Membrane tethering may provide a way to reduce degradation of unstable proteins. Degradation of misfolded proteins is a key mechanism for cellular maintenance and a major use of the ubiquitin modification system. Misfolded cytoplasmic proteins undergo polyubiquitination with K48-linked chains; these are then recognised by the proteasome and the ubiquitinated proteins degraded The key common requirement is that misfolded proteins, in various locations, are recognised as such by an appropriate ubiquitin ligase. In yeast, several have been identified. For ER-associated degradation the membrane-associated RING domain E3 ligases Hrd1 and Doa10 are the prime candidates Despite these findings, there remains considerable uncertainty over the systems responsible for degradation of abnormal proteins, partly because of redundancy. For example, soluble proteins may aggregate and undergo autophagy as well as being degraded by the proteasome We have sought to understand the full range of quality control of membrane proteins. Though most mistakes occur during the initial folding process, and thus can be detected at the level of the ER or Golgi, there is at least the potential for damage to proteins after they arrive at their destination. Are plasma membrane proteins constantly monitored for damage, and if so, how are they disposed of? If, for example, the cytoplasmic domain of a plasma membrane protein is damaged, will it be recognised by the cytoplasmic proteasomal system, or targeted to the vacuole? To address this, we have fused temperature sensitive soluble proteins to membrane anchors and investigated their degradation after a temperature shift. Surprisingly, though these proteins are degraded when soluble or attached to the ER, once delivered to the cell surface or vacuolar membrane they are much more stable. It appears that such proteins are quite well tolerated by cells, and there is no quality control system designed to destroy them rapidly.cim3-1  in doa10 mutant  was efficiently degraded in a e mutant . It appeHaving established that the ts alleles of Ura3 could induce degradation of attached proteins, we fused the mutant Ura3-GFP chimeras to the plasma membrane SNARE protein Sso1. Fluorescence microscopy confirmed that the bulk of the Ura3-2(GFP-Sso1) construct was indeed at the plasma membrane . Cells eWe extended these studies by fusing Ura3-2(GFP) to three more SNARES: Snc1, also found mainly on the plasma membrane; Vam3, found on the vacuolar membrane; and Use1, an ER resident SNARE. The Snc1 and Vam3 fusions also avoided degradation after temperature shift . In contcim3-1 proteasome mutant, but was degraded in cells lacking Doa10, San1 or both  of the N-terminal 92 amino-acid region of the bacteriophage lambda repressor. The A66T mutation is present in the c1857 temperature-sensitive repressor  or both . Thus, iDespite these differences between the properties of the lambda repressor and Ura3 mutants, fusion of the repressor-GFP construct to Sso1 inhibited its degradation, just as with the Ura3 mutants . HoweverIt has been reported that some abnormal proteins, such as a temperature sensitive mutant of Ubc9, accumulate in discrete punctae prior to degradation GAL1 promoter to drive synthesis of Ura3-2(GFP) at low temperature, added glucose to repress transcription for four hours, then subjected the cells to high temperature. The Ura3-2(GFP) construct was quite uniformly distributed at 25°, with only an occasional bright spot . HoweverWe also examined the GFP-tagged lambda repressor mutant, and found that it had a dispersed distribution with a few small bright dots, whether the cells were kept at 16° or had been shifted to 30° for one hour . Taken tOur goal was to test how yeast cells deal with membrane proteins whose cytoplasmic domain becomes damaged. Using two temperature-sensitive mutants of Ura3 and one of the lambda repressor, all of which are degraded at high temperature when in soluble form, we reached the surprising conclusion that plasma or vacuolar membrane proteins bearing misfolded domains are not rapidly degraded either by the proteasome system or the vacuolar system. However, when attached to the ER, the domains do trigger rapid proteasomal destruction. It seems that cells rely on the ERAD system to catch misfolded proteins before they escape to other organelles, and have no special way to remove them if they misfold after reaching their destination.It seems likely that the most common problem with membrane proteins is a failure to fold initially, in which case they are subject to ERAD. It may be that misfolding at a later stage is uncommon, and since the proteins we have studied do not aggregate when membrane-tethered, they may not be particularly harmful to the cell. Perhaps, therefore, there is no need for machinery to remove them.What is curious, however, is that the proteins are not degraded even though they are exposed to the cytosol, and when free to move in the cytosol are good substrates for degradation. We considered several possible explanations for this. One is that some form of aggregation or localisation is necessary for degradation, but is prevented by membrane tethering. However, we could find little evidence that degradation of the soluble forms of the proteins was linked to their localisation. Second, the membrane proteins may be inaccessible to the ubiquitin ligases that recognise unfolded proteins. This is a plausible explanation, since the Ura3 mutants required either Doa10, an ER membrane protein, or San1, a nuclear protein, for their degradation. These ligases do seem to be major contributors to quality control, since several nuclear temperature-sensitive proteins are San1 substrates A third possibility is that the location next to a membrane is a protected environment, from which ubiquitin ligases and/or proteasomes are excluded, except for the ER where specific targeting mechanisms overcome this. It is notable that specific machinery is thought to recruit proteasomes to the ER membrane as well as extract membrane proteins It is also possible that quality control at the plasma membrane is actively countered, for example by localised chaperones or deubiquitinating enzymes. Deubiquitination has in fact been proposed to explain why the Pma1-7 mutant of the plasma membrane ATPase, which is normally ubiquitinated and routed from Golgi to vacuole, appears stable if allowed to reach the plasma membrane It may be that the unfolded proteins are ubiquitinated on the plasma membrane, but escape degradation simply because they cannot be extracted from this membrane and are poor substrates for endocytosis. It could be, for example, that K48-linked ubiquitin chains are not well recognised by the endocytic machinery and instead are removed by deubiquitinating enzymes. If so, one might expect ubiquinated forms of the ts proteins to appear after temperature shift. We did in fact detect ubiquitinated forms of the ts Ura3(GFP-Sso1) constructs. However, the appearance of these forms was not reproducibly temperature-dependent, and they represented a very minor fraction of the protein. We were therefore unable to rule out the alternative possibility that they comprised a minor fraction of constitutively misfolded, and thus non-fluorescent, protein that remains in the ER and undergoes ERAD. The question of ubiquitination at the plasma membrane is thus still open.It remains to be seen whether the yeast results can be generalised to other species, but there are reasons to be cautious. For example, misfolded proteins are usually recognised by chaperone proteins, and in animal cells persistently misfolded proteins are thought to become substrates for a soluble chaperone-associated ubiquitin ligase termed CHIP It is possible, of course, that machinery exists in yeast to remove plasma membrane proteins with cytoplasmic domains that are more extensively damaged than is the case for the three ts proteins we have used, for example if they undergo extensive aggregation. Indeed, the temperature sensitive Pma1-10 protein has been reported to be degraded after arrival at the cell surface MATα uraΔ0 leu2Δ0 his3Δ1 lys2Δ0) background. The doa10 san1 double mutant was made by homologous recombination with a replacement of the DOA10 open reading frame in the BY4742 san1 knockout strain by a PCR product encoding the Schizosaccharomyces pombe HIS5 gene. The doa10 hrd1 double mutant was made similarly. The cim3-1 and corresponding wild type strain Yeast strains unless otherwise stated were from the Open Biosystems knockout collection in a BY4742  together with Nco1-Bpu101 cut pmUra414 and selected on plates lacking tryptophan. A pool of colonies was then plated onto 5-fluoroorotic acid plates at either 25°C or 37°C. Colonies from these plates were streaked out and further tested for temperature sensitive growth on plates lacking uracil. Rescued plasmids were sequenced.PmUra414 was made by insertion of a c-myc tagged PCR product from the SNC1 and VAM3. USE1 was cloned by PCR into a TPI1-promoter-GFP containing version of pRS416 and the GFP-USE1 fusion used to replace GFP in the URA3 fusion constructs.In-frame GFP fusions were made between downstream EcoR1 and BamH1 sites. The SSO1 fusion was made by replacement of the GFP with a GFP-SSO1 fusion URA3 moiety in the GFP fusion constructs. Galactose-inducible constructs were made by placing the fusion constructs in a version of YCpLac33 carrying the GAL1/10 promoter fragment. Sequences of all PCR generated constructs were verified by DNA sequencing.The region encoding the first 92 amino acids from the c1857 bacteriophage lambda repressor was cloned by PCR and subsequently mutated by PCR to introduce a L57A change cim3-1 strain, cells were preincubated for 1 hour at 37°C prior to the addition of cycloheximide. For the galactose shutoff experiments cells were transferred from medium containing 2% galactose and raffinose into medium containing 2% glucose for 4 hours before temperature shift.Cells were grown into early log phase and after addition of cycloheximide (to 500 µg/ml) were shaken at the temperatures shown, and samples taken at the indicated times. These were spun down and stored on dry ice before being subjected to alkaline lysis and solublisation as described by Live cell imaging was performed on concanavalin-A coated slides using a Zeiss LSM510. Images were adjusted for contrast and brightness, and in some cases, they were blurred to filter noise, by using Adobe Photoshop .


Despite extensive research, the details of the biological mechanisms by which cancer cells acquire motility and invasiveness are largely unknown. This study identifies an invasion associated gene signature shedding light on these mechanisms.We analyze data from multiple cancers using a novel computational method identifying sets of genes whose coordinated overexpression indicates the presence of a particular phenotype, in this case high-stage cancer.COL11A1 and other genes, mainly THBS2 and INHBA. The composition of the overexpressed genes indicates invasion-facilitating altered proteolysis in the extracellular matrix. The prominent presence in the signature of INHBA in all cancers strongly suggests a biological mechanism centered on activin A induced TGF-β signaling, because activin A is a member of the TGF-β superfamily consisting of an INHBA homodimer. Furthermore, we establish that the signature is predictive of neoadjuvant therapy response in at least one breast cancer data set.We conclude that there is one shared "core" metastasis-associated gene expression signature corresponding to a specific variant of stromal desmoplastic reaction, present in a large subset of samples that have exceeded a threshold of invasive transition specific to each cancer, indicating that the corresponding biological mechanism is triggered at that point. For example this threshold is reached at stage IIIc in ovarian cancer and at stage II in colorectal cancer. Therefore, its presence indicates that the corresponding stage has been reached. It has several features, such as coordinated overexpression of particular collagens, mainly Therefore, these results can be used for developing high specificity biomarkers sensing cancer invasion and predicting response to neoadjuvant therapy, as well as potential multi-cancer metastasis inhibiting therapeutics targeting the corresponding biological mechanism. There is currently great interest in identifying the biological mechanisms for the acquisition of motility and invasiveness in cancer. It has been hypothesized ,2 that t-6), leading to the hypothesis that coordinated overexpression of these genes implies that the tumor has progressed to at least stage IIIc.A study  of serouTo further validate this hypothesis and test if similar versions apply to other cancers, we developed a computational technique identifying in an unbiased manner coordinatedly overexpressed genes associated with a phenotype (such as transition to a particular stage). Our results consistently rediscover the same "core" signature of overexpressed genes, confirming the hypothesis. We found that the signature is present in multiple cancers, each of which has its own features involving additional genes, but the core signature is common.FAP (fibroblast activation protein) and ACTA2  in the set of overexpressed genes, the signature suggests a variant of stromal desmoplastic reaction. As further evidence, it is known FAP  phenotype that naturally fits this description, ignoring the expression levels of the genes outside the region of overexpression, which we call "extreme value association" (EVA). The same measure can identify coordinately silenced genes, but we did not find any such genes across various cancer types.M samples, out of which N are "low stage" and M - N are "high stage," and we select the m samples with the highest gene expression values, out of which n are "low stage" and m - n are "high stage." Under the assumption that gene expression values are uncorrelated with the phenotype, the probability that there will be at most n "low stage" samples among the selected m samples is given by the cumulative hypergeometric probability h. The EVA metric is then defined as equal to -log10 of the minimum of these probabilities over all possible choices of m, in which m ranges from 1 to M (note that n depends on m). For example, assume that there are 250 high-stage samples and 50 low-stage samples for a total of 300 samples. Furthermore, assume that the 100 samples with the highest values of a particular gene contain 99 high-stage samples and one low stage sample. In that case, h can be evaluated using the MATLAB function hypercdf  = 5 × 10-9, resulting in the EVA metric for that gene of at least -log10(5 × 10-9) = 8.3. If the 101th sample is also high-stage, then the EVA metric of the gene will be even higher. Note that, once the highest value is reached, the sorting arrangement of the remaining samples is irrelevant, reflecting the hypothesis that only the extreme values are associated with the phenotype. Figure COL11A1 gene using the TCGA ovarian cancer data set and the staging threshold for the definition of the binary phenotype set between IIIb and IIIc. The maximum (8.31) occurs when m = 133.The EVA metric is the minimum p-value of biased partitions over all subsets of samples with highest expression values of the gene. In other words, suppose that there are totally -3 after Bonferroni correction. Finally, we find the intersection of these selected gene sets over all cancer expression data sets and rank them in terms of fold change.We then developed a mechanistic and unbiased (only dependent on the phenotype) algorithm, which, when given a gene expression data set for a number of samples labeled "high stage" or "low stage," leads to a selection of genes that are coordinatedly overexpressed only in high-stage samples, ignoring the effect of the rest, thus precisely reflecting the observed phenomenon. We first select the top 100 genes that rank highest according to the EVA metric criterion using a mixture analysis  of both overexpressed and silenced genes. Using this set of genes only, we perform k-means clustering with gap statistic . At thatn samples and m genes, the EVA algorithm computes cumulative hypergeometric distribution probabilities nm times. This can be computationally intensive, so we devised a low-complexity implementation algorithm described in Additional file For a data set with G1 and G2, are governed by a joint probability density p12 with corresponding marginals p1 and p2 and using simplified notation, the mutual information I is a general measure of correlation and is defined as the expected value G1, G2, with respect to a third variable G3 is [I - [I + I], i.e., the part of the association of the pair G1, G2 with G3 that is purely due to a synergistic cooperation between G1 and G2 (the "whole" minus the sum of the "parts"). We used a 3rd order B-spline-based mutual information estimator dividing the observation space into six equally spaced bins in each dimension.Assuming that two variables, such as the expression levels of two genes le G3 is  equal toF. The p-value of an actual score x0 is then 1 - F(x0) under the null hypothesis of no association with phenotype. Similarly, for the synergistic pair, we found the top-scoring synergy in 100 data sets that were identical to the original except that the phenotype values were randomly permuted on each, and the top permuted synergy scores were modeled, as above, with the Gumbel distribution.We applied a permutation-based approach accounting for multiple test correction: We did 100 permutation experiments of the class labels, saving the corresponding 100 highest values after doing exhaustive search in each permutation experiment. Using the set of these 100 highest value scores, we obtained the maximum likelihood estimates of the location parameter and the scale parameter of the Gumbel  distribution, resulting in a cumulative density function We performed the EVA algorithm on four rich gene expression data sets, two from ovarian cancer, the one from TCGA and another one , and two-3 in all four data sets. Table COL11A1 (probe 37892_at). Again, these genes were found purely as a result of their association with the staging phenotype in all four cancers. Gene Ontology enrichment testing of these genes identified cell adhesion, extracellular matrix and collagen fibril organization. It turns out that use of other standard correlation measures instead of the EVA measure in the same algorithms leads to the same results, because the overall correlation of these genes with the phenotype is strong merely as a result of the genes' overexpression in some high-stage samples alone. The EVA method has the additional advantage of providing an estimate of the size of that subset of high-stage samples.Remarkably, we found that there were 137 genes  with log fold change of 6.50, while the next highest (4.08) corresponded to another probe of COL11A1, followed by a probe of COL10A1. Second, a study [COL11A1 (probe 37892_at) was at the top (log fold change: 4.26) followed by COL10A1 and FAP. Third, a study of pancreatic ductal adenocarcinoma [COL11A1 (probe 37892_at) is again prominent and the top entry (log fold change 5.15) was INHBA, supportive of our hypothesis of activin A induced TGF-β signaling. The presence of the MAF signature in the latter study indicates that pancreatic cancer had already become invasive in most cases before the biopsy. The prominent desmoplastic reaction in pancreatic cancers (which contains the MAF signature) has recently been increasingly recognized as a "foe" [COL11A1 has been identified as a potential metastasis-associated gene in other types of cancer as well, such as in lung [Specifically, a breast cancer study  comparin a study  comparinarcinoma  identifi a "foe"  that cou in lung , and ora in lung , suggestCOL11A1 (probe 37892_at). This remarkable consistent strong association of COL11A1 with the staging phenotype (specific to each cancer type) suggests that it could be used as a "proxy" of the MAF signature. This would allow us to improve on the gene list of Table In those cases as well as in our own findings, there was prominent presence of COL11A1. Additional file COL11A1. The list is similar to the phenotype-based gene ranking (Table THBS2), inhibin beta A (INHBA), leucine rich repeat containing 15 (LRRC15), versican (VCAN), fibroblast activation protein (FAP), and matrix metallopeptidase 11 (MMP11) aka stromelysin 3. The presence of FAP indicates a general desmoplastic reaction and is not, by itself, sufficient for inferring the MAF signature.As a first step for this task, we identified the genes that are consistently highest associated with ng Table . In addiCOL11A1 was uniquely not associated with any of these genes in noncancerous samples, further supporting the hypothesis that it can be used as a proxy for the MAF signature. Our results indicate that THBS2 and INHBA, top ranked in Table Furthermore, contrary to all other genes, COL11A1 jointly, but not individually, and therefore they would not appear in the previous list. For this task we ranked gene pairs according to their synergy [COL11A1, using the computational method in [ECM2 and TCF21 are jointly, but not individually, strongly associated with COL11A1  in the two ovarian cancer data sets. Such findings are useful for developing biological hypotheses, e.g. in this particular case they suggest that in ovarian cancer the extracellular matrix protein 2 is associated with the MAF signature only when the TCF21 gene  is downregulated.As a second step, we identified gene pairs that are highest associated with  synergy  with COLethod in , which c-4), based on the genes highest associated with COL11A1.The MAF signature exists even in non-epithelial cancers. Indeed, we confirmed that neuroblastoma also carries the MAF signature consistently associated with high stage: As shown in Additional file COL11A1, we found many statistically significant miRNAs, among them hsa-miR-22 and hsa-miR-152, as well as differentially methylated genes, such as SNAI1 and PRAME, suggesting a particularly complex biological mechanism . Table -16 in both cases, see Methods). SNAI1 (snail) methylation is particularly important as the gene is known as one of the most important EMT-related transcription factors. Instead, the strongest MAF-associated transcription factor is AEBP1. Many of the other EMT-related transcription factors, such as SNAI2, TWIST1, and ZEB1 are often overexpressed in the MAF signature, but SNAI1 is not . We believe that the lack of SNAI1 expression is a key distinguishing feature of the MAF signature, in which we observed neither SNAI1 overexpression nor CDH1 (E-cadherin) downregulation, at least on the mRNA level.We only had miRNA and methylation data available for the TCGA ovarian data set. Using as measure the mutual information with DCN (decorin). Although some of the MAF key genes (such as COL11A1 and THBS2) were not among these 50, the metagene signature used in that study has a significant intersection with the MAF signature and was found resistant to neoadjuvant chemotherapy. To compare the drug response performance of the DCN metagene set with that of the MAF signature, we used the top 50 genes of the MAF signature in terms of their association with COL11A1 from various cancers  more than high expression of the DCN metagene set (identifying 12 samples out of which 10 have lack of response), suggesting that the presence of the core genes of the MAF signature provide at least as good indicator of resistance to neoadjuvant chemotherapy. The reason for the drug resistance may simply be that the invasiveness sensed by the MAF signature does not allow the size or extent of the cancer to be reduced prior to surgery.Significantly, we also found that, at least in ER negative breast cancer, the MAF signature is associated with resistance to neoadjuvant FEC. This was demonstrated in  where a COL11A1, INHBA, and THBS2, as shown in the scatter plots of Figure A direct clinical application of these findings is the development of a high-specificity invasion-sensing biomarker product detecting coordinated overexpression of a few top-ranked genes, such as LRRC15 (aka LIB) appears important but unclear, though it has already been recognized as promoting migration through the extracellular matrix [Of course, the most significant clinical application would be to develop metastasis-inhibiting therapeutics using targets deduced from the biological knowledge provided by the MAF signature. Our top ranked genes strongly suggest that they are produced by myofibroblasts or myofibroblast-like cells activated by activin A-induced TGF-β signaling and leading to some form of altered proteolysis , which rr matrix . VersicaAlthough each of the MAF signature molecules could serve as a potential therapeutic target, the hypothesis that activin A signaling is at the heart of the MAF mechanism immediately suggests that follistatin (activin-binding protein) could serve as a metastasis inhibitor, which is exactly what recent research  indicatein situ and stage I, the signature would not be apparent. Second, each cancer type may have its own additional features accompanying the MAF signature. For example, in ovarian cancer it is accompanied by sharp downregulation of genes COLEC11, PEG3 and TSPAN8, which is not the case in other cancers. Indeed, the main contribution of our work is the identification of the common multi-cancer "core" signature, from which a universal metastasis-associated biological mechanism can be identified. Third, the MAF signature may be reversible, perhaps as a result of the disappearance of many of the stromal cells in the mature desmoplastic stroma when it is replaced by "acellular" matrix [There are several reasons that the core MAF signature has not yet been discovered as a multi-cancer metastasis-associated signature. First, it is essential to identify the correct phenotypic staging threshold recognizing that the signature only exists in a subset of tumors that exceed that particular stage. Indeed, if the threshold in breast cancer was put between stage I and stage II, or between stage II and stage III, rather than between " matrix . The preAn important topic of further research is the determination of the precise biological event of interaction of cancer cells with the microenvironment that gives rise to the stromal MAF signature and associated invasiveness. Because of the recognized similarities with the mechanisms of wound healing , it is lSeveral of the top-ranked genes in the MAF signature  are known to be potent anti-angiogenesis mediators. The reversal of the MAF signature would thus facilitate further metastatic dissemination to distant sites. In other words, (a) the desmoplastic MAF signature and (b) angiogenesis, are two independent biological events. The former appears to be based on activin A signaling, as several of the MAF proteins in addition to INHBA are also known inhibitors of the standard TGF-β ligand. The reversal of the MAF signature would allow the standard ligand to take over in TGF-β signaling, and may thus facilitate further metastasis. These observations provide explanations for the seemingly contradictory observed roles of TGF-β signaling inhibiting early cancer but facilitating metastasis.The possible reversibility of the MAF signature leads to the intriguing hypothesis that perhaps all metastases have, at some point temporarily been there, which explains why we only observe it in a subset of them. This would be particularly exciting, because in that case any metastasis-inhibiting therapeutic intervention targeting the MAF mechanism would be widely applicable to low-stage tumors.In conclusion, we have shown that, using purely computational analysis of publicly available biological information, systems biology has revealed the core of a multi-cancer metastasis-associated gene expression signature. In the near future, a vast amount of additional information will become available, including next generation sequencing, miRNA and methylation information for many cancers, which will allow additional computational research building on this work and clarifying the details of the underlying invasion-associated complex biological process.The authors declare that they have no competing interests.DA and VV conceived of and supervised the study. HK and JW carried out the experiments. DA drafted the manuscript. All authors participated in the study, read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1755-8794/3/51/prepubHeat map of TCGA ovarian cancer data set. This file contains the result of hierarchical clustering for the TCGA ovarian cancer data set using a particular gene set (see text).Click here for fileImplementation of the EVA algorithm. This file contains the description of a low-complexity implementation of the EVA algorithm.Click here for fileList of genes resulting from EVA algorithm. This file contains the commonly significant probe sets in four ovarian and colorectal cancers data sets.Click here for fileListing of genes in 9 cancer data sets. This file contains a color-coded list of genes in various cancers ranked in terms of the mutual information with COL11A1.Click here for fileHeat map of neuroblastoma data set. This file contains the result of hierarchical clustering for the neuroblastoma data set (GSE3960) using the MAF signature genes.Click here for fileHeat map of breast cancer data set using MAF signature genes. This file contains the result of hierarchical clustering for the breast cancer data set (GSE4779) using the MAF signature genes.Click here for fileDCN metagene setHeat map of breast cancer data set using . This file contains the result of hierarchical clustering for the breast cancer data set (GSE4779) using the DCN metagene set.Click here for file


Standard approach to the treatment of head and neck cancer include surgery, chemotherapy, and radiation. More recently, dramatic increases in our knowledge of the molecular and genetic basis of cancer combined with advances in technology have resulted in novel molecular therapies for this disease. In particular, gene therapy, which involves the transfer of genetic material to cells to produce a therapeutic effect, has become a promising approach. Clinical trials concerning gene therapy strategies in head and neck cancer as well as combination of these strategies with chemotherapy and radiation therapy will be discussed. The prognosis of patients with Squamous Cell Carcinoma of the Head and Neck (SCCHN) is poor. The National Cancer Institute (NCI) reported in its SEER Cancer Statistic Review that there are approximately 40,000 new cases of primary head and neck cancer in the United States each year. The incidence of this cancer has been gradually increasing over the past 20 years and it is now the fifth leading cause of cancer incidence and the sixth leading cause of cancer-related death in the world . PatientGene therapy involves the introduction of foreign DNA into somatic cells to produce a therapeutic effect . A variep53 DNA throughout the manufacturing process. The p53 gene is located on chromosome 17p in humans and it encodes a 393 amino acid protein that is critical to tumor biology [Advexin  is an adenovirus (type 5) in which the E1 region is replaced with the cDNA of the p53 gene and is driven by a cytomegalovirus (CMV) promoter [ biology . InactivA phase I trial carried out at the MD Anderson Cancer Center, Texas, USA, in patients with advanced local or regional head and neck cancer that was unresectable was reported by Clayman ,6. ThirtTwo phase II trials (T-201 and T-202) using this vector (ING-201) have been concluded at many centers and early results in 170 patients receiving intratumoral injection of Ad-p53 over a variety of doses and schedules showed that treatment with ING-201 is safe and effective [There are two ongoing phase III trials to compare the safety, efficacy and overall survival of treatment with Advexin as monotherapy or in combination with chemotherapy in patients with head and neck cancer. In one of these trials (T301), patients with local or regional recurrent refractory SCCHN who have failed radiation therapy and chemotherapy with platinum-containing drugs or taxanes were randomized to either Advexin intratumoral or methotrexate intravenously. Patients were treated for a maximum of nine cycles (27-weeks). Survival was the primary end point of the study and predicted accrual to accomplish this endpoint was 240 patients. In the second study (T302), patients with local or regional recurrent SCCHN who have not been previously exposed to chemotherapy were randomized to receive a combination of intratumoral Advexin, cisplatin and 5-fluorouracil (5-FU) or standard care with cisplatin/5-FU. The primary end point was time to progression and predicted accrual to accomplish this endpoint was 288 patients.Gendicine is another different molecular entity that combines an Adenovirus type 5 vector with a p53 expression cassette, using the RSV promoter and BGH (A) tail .In a Phase I trial using the adenoviral vector SCH-58500, 16 patients with HNSCC received escalated doses ranging from 7.5 × 10^9 PFU to 7.5 × 10^12 PFU. Toxicity was limited to grade 1–2 fever and injection pain. One patient achieved a partial remission (PR), which correlated with the induction of apoptosis and transgene expression [Recently, the first randomized clinical trial of p53 gene therapy was reported. Ninety patients with SCCHN were randomly allocated to receive either intratumoral injections of Ad-p53 in combination with radiation therapy (70 Gy/8 weeks) or radiation therapy alone. Complete remission was seen in 64.7% of patients receiving Ad-p53 combined with radiation therapy compared with 20% of patients receiving radiation therapy alone, which was statistically highly significant . This clONYX is a replication-conditional adenovirus that is defective in the early regulator protein E1B, which binds to and inactivates p53 to promote its own activation . Cells cA dose-escalation phase I trial was carried out by Ganly . A totalA subsequent phase II trial showed enhanced efficacy when the virus was given by multiple daily injections in the same group of patients ,20. In tThe viral construct DL1520 has also been employed in chemoprevention as a mouthwash preparation for patients with oral leucoplachia and displasia . 19 asseTwo interesting Phase II clinical trials have showed the feasibility and the preliminary efficacy of ONYX in association with chemotherapy ,23. BothH-101 is an E1B-55k and partly E3 deleted adenoviral vector comparable to ONYX-015. In a randomized phase III clinical trial of H-101 in combination with cisplatin and 5-FU, virus particles were administered by intratumoral injection daily for five consecutive days every three weeks . A respoThe E1A gene functions as a tumor inhibitor by repressing oncoproteins and sensitizing cancer cells to chemotherapeutic and radiation treatments. The interaction of E1A, a nuclear phosphoprotein with a wide range of cellular proteins in multiple signal transduction pathways , results in multiple biological activities. E1A was initially appreciated for its ability to repress transcription, leading to down regulation of HER-2/neu protein and resulting in loss of malignant phenotypes. The anti-oncogenic activity of the E1A gene, however, is not limited to tumors that overexpress HER-2/neu. E1A also modulates expression of other genes, resulting in differentiation of certain cancer cells. It has also been shown to enhance antitumor activity in response to VP16, cisplatin, paclitaxel, and adriamycin. Several studies have also demonstrated a significant tumor radiosensitisation response to E1A therapy. Clinical investigations using cationic liposomes mixed with plasmid DNA encoding for E1A have shown safety and efficacy in animal models, as well as preliminary safety and activity in clinical trials. In one small pilot study, nine patients with recurrent, unresectable breast cancer and nine patients with recurrent, unresectable HNSCC, the E1A gene was administered using a lipid complex. No toxic effects were observed and the highest dose of drug treatment that does not cause unacceptable side effects (MTD) was not reached . In a suIn 1 trial, 9 patients with advanced SCCHN who did not express the HLA-B7 antigen received intratumoral injection of an HLA-B7 plasmid (Allovectin-7). No toxic effects were observed. Four of 9 patients achieved a partial response and induction of HLA-B7 expression was confirmed in 2 of 4 patients who responded . On the The literature is full of preclinical studies describing gene therapy products employed as anticancer agents. However only a few of these approaches have been tested in clinical trials. This review focuses only on gene therapy strategies applied in clinical setting in head and neck cancer patients where the results of clinical trials have been reported.Several important observations can be made from the clinical trials performed to date. Non-replicating and conditional replicating adenoviral vector are the most interesting gene therapy strategies explored in HNSCC. Intratumoral administration of these products is easy and possible, since these tumors are often accessible for direct injection.As anticancer agents, viral products have an excellent profile and do not appear to enhance the toxicity of either chemotherapy or radiation making them good candidates for combined modality treatment strategies.The most frequently reported adverse events with adenoviral products are fever and chill, asthenia, injection site pain, nausea and vomiting. However, the vast majority of these adverse events are mild to moderate.Efficacy is limited to loco-regional control. In head and neck cancer, local and/or regional tumor recurrence develops in approximately one-third of patients, despite definitive treatment. Two-thirds of patients dying of this disease have no evidence of symptomatic distant metastases. Therefore, local and regional disease control is paramount in this disease, underscoring an urgent need for more effective local therapies. The promise of adenovirus-mediated gene therapy has not yet translated in patient survival primarily because of the inability to deliver the therapeutic gene to a large number of cells. Further optimization of vectors will be essential for the improvement of clinical effectiveness of cancer gene therapy.Although preliminary clinical data are encouraging, well-designed studies are needed for evaluation of these new anticancer agents before their approval in clinical practice. Approval of adenoviral products in China for treatment of SCCHN has been subject of many controversies in the USA. Approval should be based on multi-center, multinational randomized studies planned to follow up patients in order to evaluate survival endpoint. Moreover, correlative endpoints, including assessment of expression of the transferred genes, immunologic response to transgenes and vectors, vector kinetics and verification of viral replication, if replicating vectors are employed, are crucial in order to validate the clinical utility of a given approach in combination with more traditional endpoint such as safety and efficacy.Lastly, because gene therapy involves introducing changes to the body's set of genetic instructions, there are some important ethical issues that need to be considered on the use of gene therapy. Besides the classical concerns about the cost of gene therapy and availability to the general population, there are some safety issues that need to be considered. Recently, it has been described that somatic gene therapy resulted in an inheritable change to the genome of rats . This phThe authors declare that they have no competing interests.MV and PPC discussed the manuscript topics and drafted the manuscript.


Bile acids, end products of the pathway for cholesterol elimination, are required for dietary lipid and fat-soluble vitamin absorption and maintain the balance between cholesterol synthesis in the liver and cholesterol excretion. They are composed of a steroid structure and are primarily made in the liver by the oxidation of cholesterol. Cholesterol is also highly abundant in the human ovarian follicle, where it is used in the formation of the sex steroids.Here we describe for the first time evidence that all aspects of the bile acid synthesis pathway are present in the human ovarian follicle, including the enzymes in both the classical and alternative pathways, the nuclear receptors known to regulate the pathway, and the end product bile acids. Furthermore, we provide functional evidence that bile acids are produced by the human follicular granulosa cells in response to cholesterol presence in the culture media.These findings establish a novel pathway present in the human ovarian follicle that has the capacity to compete directly with sex steroid synthesis. Bile acids are powerful detergents with essential functions in lipid and cholesterol processing. They play key roles in the gastrointestinal and hepatobiliary systems, are required for dietary lipid and fat-soluble vitamin absorption, and maintain the balance between cholesterol synthesis in the liver and cholesterol excretion Bile acid synthesis can occur through two pathways: the classic  pathway or the alternative (acidic) pathway There are well-known feedback loops controlling gene transcription of the major enzymes in bile acid synthesis, especially CYP7A1 and CYP8B1 In addition to its presence in the gastrointestinal and hepatobiliary systems, cholesterol is known to be highly abundant in the human ovarian follicle, where it is used in the formation of the sex steroids Here we describe for the first time evidence that all aspects of the bile acid synthesis pathway are present in the human ovarian follicle, including the enzymes in both the classical and alternative pathways, the nuclear receptors known to regulate the pathway, and the end product bile acids. Furthermore, we provide functional evidence that bile acids are produced by the human follicular granulosa cells in response to cholesterol presence in the culture media. These findings establish a novel pathway present in the human ovarian follicle that has the capacity to compete directly with sex steroid synthesis.in vitro fertilization (IVF). The individual FF samples were analyzed in duplicate for content of total bile acids by enzymatic assay. The mean bile acid concentration in FF was measured to be 17.301 µM (STD ± 5.41) (Follicular fluid (FF) from mature follicles from 93 patients was obtained during  ± 5.41) . The ori ± 5.41) . This dr ± 5.41) . The meaThis is the first demonstration of the presence of bile acids in this compartment. The high cholesterol concentration in the follicle is likely to be supporting not only active steroidogenesis but also synthesis of bile acids. This finding raises the obvious question of whether the enzymes responsible for bile acid synthesis are present in the ovarian follicle. The intrafollicular synthesis of bile acids could be facilitated by activities within the granulosa cells, the oocyte, or both.Initially, we applied gene chip array to search for the presence of mRNA transcripts of these enzymes in granulosa cells from 10 patients. The assay identified the presence of mRNA for the key enzymes in the classical and the alternative pathways of bile acid synthesis: CYP7A1, CYP27A1, CYP7B1, and CYP8B1 . Next, tIt is possible that the oocytes which reside within the ovarian follicle also participate in the synthesis of bile acids. We analyzed discarded, non-fertilized human oocytes by IF with specific antibodies for the key enzymes in both bile acid synthesis pathways. We identified specific positive staining of CYP7A1, CYP7B1, and CYP8B1 , panel CTo test for bile acids synthesis in granulosa cells we cultured primary human cumulus granulosa cells (CGC) and exposed them to media containing different cholesterol concentrations  loaded in human low density lipoprotein (LDL) particles. After 48 hours culture medium and cells have been analyzed for presence of bile acids at time 0 and time 48 hours. The experiment was repeated in triplicate. We measured a CGC-specific dose-dependent increase in production of bile acids in response to increasing cholesterol in CGC media . The resThe bile acid synthesis pathway is known to be regulated by nuclear receptors. We examined primary human granulosa cells and discarded, non-fertilized oocytes for presence of FXR, RXRα, LXRα, and LRH-1 by protein-specific antibodies and IF. FXR, which is known to have as its ligand bile acids, was found to be present in the cytosol of granulosa cells , panel BOur results strongly argue for the presence of all of the components of the bile acid synthesis pathway, including the key enzymes in bile acid synthesis as well as the receptors that regulate expression of these enzymes, and the end product bile acids. To our knowledge, this is the first report of this pathway in the human ovary.Although it is widely accepted that the classical pathway of bile acid synthesis is present only in the liver, our data clearly suggests that the key enzymes from both the classical and alternative synthesis pathways are present in the ovarian granulosa cells and oocytes. To our knowledge, these findings are the first demonstration for presence of CYP7A1, CYP27A1, CYP7B1, and CYP8B1 at both mRNA and protein level in granulosa cells. This is also the first demonstration of the presence of these enzymes in the oocytes as proteins. CYP7A1 is considered to be a liver-specific enzyme, although it has been reported to be present in human prostate Our results suggest that granulosa cells have the key enzymes to synthesize bile acids from cholesterol, and that this is an active functional pathway. The balance between cholesterol use in steroidogenesis and degradation in the bile acid pathway is not known, but most likely there is a fine balance which may prove crucial to oocyte maturation. In support of this hypothesis are recently published experimental data with rodents where a role for LRH-1 in steroidogenesis, gonadal development, aromatase expression, and progesterone biosynthesis was clearly documented Given that the ovarian follicle is a highly privileged environment, it is likely that bile acids play a specific role beyond simply acting as detergents and carriers of cholesterol. Full characterization of the follicular-specific role of bile acids remains to be determined.Follicular fluid, granulosa cells and blood serum were obtained under Beth Israel Deaconess Medical Center (BIDMC) IRB approval (Protocol #2003-P-000289) after appropriate written informed consent from women undergoing in IVF. Discarded, non-fertilized metaphase II human oocytes were obtained under BIDMC IRB approval with waiver of informed consent from IVF cycles in which oocytes failed to fertilize and would have otherwise been discarded. Waiver of informed consent for discarded, non-fertilized oocytes was granted by BIDMC IRB because no patient-identifying information was obtained with the pooled, anonymous samples of discarded tissue, and no protected health information was used.All follicular fluid from large (mean diameter >16mm) follicles, granulosa cells and blood plasma retrievals have been performed at the Boston IVF Surgery Center of Waltham, MA as previously described Total bile acid concentration in follicular fluid and cell culture media was determined using the BioQuant Total Bile Acids Assay Kit  as recommended by the supplier. All assays have been repeated in duplicate. Statistical comparisons were made using Microsoft Office Excel 2003 SP2 version 5.1.2600  with p value <0.05 considered statistically significant.Standard technique has been used for western blotting and immunofluorescence Primary granulosa cells (passage 4 or less) have been cultured in six wells plates to 70% confluence in DMEM, 10% fetal calf serum (FCS), 2.5 ng/mL bFGF (Sigma Aldrich), and 25 µg/mL Amphotericin B. Cholesterol (Sigma) was added to the cell culture medium as a LDL complex in the following concentrations: 13.95mg/dL, 26.09mg/dL, 46.15mg/dL. After 48 hours incubation with cholesterol, the culture medium from each well has been removed and stored for cholesterol and bile acids content assay. Cells have been also collected to measure the protein content in the individual wells and use it as a base for comparison. Incubation with LDL alone without cholesterol addition has been used as negative control. All measurements have been performed in duplicates and in three independent experiments with different preparations of primary cell cultures. The cholesterol content in the culture mediums was measured by the Cholesterol Liquid Color Assay  as recommended by the supplier. BioQuant Total Bile Acids Assay Kit  was used to determine the bile acids content. After removing the culture medium the total cellular protein amount in each well was measured by the Lawry method (Bio-Rad kit) and used as a base for comparison.Pooled GC RNA from 10 patients was analyzed for gene expression profile using Agilent gene chip array. Ingenuity Systems Pathway Analysis Software  was used to interpret microchip array data.