Since the mid-1980's there has been a worldwide resurgence of severe disease from group A streptococcus (GAS), with clonal clusters implicated in Europe and the United States. However GAS associated sepsis and rheumatic fever have always remained at high levels in many less developed countries. In this context we aimed to study GAS necrotising fasciitis (NF) in a region where there are high background rates of GAS carriage and disease.We describe the epidemiology, clinical and laboratory features of 14 consecutive cases of GAS NF treated over a seven year period from tropical northern Australia.emm type 1 and two emm type 3 strains.Incidence rates of GAS NF in the Aboriginal population were up to five times those previously published from other countries. Clinical features were similar to those described elsewhere, with 7/14 (50%) bacteremic and 9/14 (64%) having associated streptococcal toxic shock syndrome. 11/14 (79%) had underlying chronic illnesses, including all four fatalities . Important laboratory differences from other series were that leukocytosis was absent in 9/14 (64%) but all had substantial lymphopenia. Sequence typing of the 14 NF-associated GAS isolates showed no clonality, with only one While NF clusters can occur from a single emergent GAS clone, this was not evident in our tropical region, where high rates of NF parallel high overall rates of GAS infection from a wide diversity of strains. The specific virulence factors of GAS strains which do cause NF and the basis of the inadequate host response in those patients who develop NF on infection with these GAS require further elucidation. Streptococcus pyogenes (Group A streptococci, GAS) alone, or in association with Staphylococcus aureus or Staphylococcus epidermidis [Necrotising fasciitis (NF) is an aggressive and rapidly destructive soft tissue infection resulting in high mortality and significant long term morbidity. Two groups of infectious agents have been described – Type I which are polymicrobial, involving anaerobic bacteria and streptococci other than serogroup A, and Type II from dermidis . The latdermidis  and has Since the mid-1980's a worldwide increase in infections due to GAS has been noted. In affluent populations where GAS disease is uncommon aside from pharyngitis in childhood, increasing numbers of necrotising fasciitis and streptococcal toxic shock syndrome (STSS) have been seen, as well as an upsurge of acute rheumatic fever apparently restricted to parts of the United States -6. This Recent case series of GAS NF have been published from Norway and OntaThe Royal Darwin Hospital services 135,000 inhabitants of the NT, with around 100,000 located in the city of Darwin. Indigenous Aboriginal Australians account for 24% of the population[Fourteen cases fulfilling the case definition were identified between May 1994 and April 2001. A chart review was conducted to identify epidemiological characteristics, clinical features, laboratory results, treatment and outcomes. Age, gender, ethnicity, urban or rural residence and any concurrent medical conditions were documented. The dates of presentation, presumptive diagnosis of NF and of surgery were documented. Symptoms, signs, hemodynamic status and temperature were documented for these dates, as was the presence of STSS as defined by the Working Group on Severe Streptococcal Infections. AntibioIncidence rates of GAS NF for this region of the NT were calculated using regional population numbers from census data. There was possibly an increase in case ascertainment after 1998 and incidence rates were also calculated by year.emm sequence typing and analysis of emm-specific PCR products were carried out as previously described[emm sequences deposited in GenBank and in the Centers for Disease Control (CDC) Streptococcus pyogenes emm sequence database[Procedures for described. DNA seq database.Approval for the study was obtained from the Health Research Ethics Committee of Royal Darwin Hospital and the Menzies School of Health Research.Table From 1994 to 2001, 14 cases of confirmed GAS NF were treated at Royal Darwin Hospital. The number of cases per year ranged from zero in 1995 and 1998 to five in 2001. This gives a maximum yearly incidence of 3.8 cases per 100,000. Half of the cases occurred in Aboriginal patients, with an incidence of 5.8 per 100,000 for Aboriginals in the 2001 cluster of cases. Eight cases occurred in women (57%), five of whom were Aboriginal. Ages ranged from 27–61 years in males (mean 41.5 yrs) and 27–59 years in females (mean 41.5 yrs). Eleven cases were community acquired and three nosocomial. All seven Aboriginal patients had multiple concurrent medical conditions, whereas three of the seven non-Aboriginals had no additional pathology.Patients presented most consistently with pain in the affected area. The other common complaints were swelling, and systemic symptoms of fever and rigors. Signs on presentation were typically induration and tense swelling of the area. Only four of the fourteen cases (29%) were noted to have extensive skin erythema, but nine (64%) had local pre-existing wounds or ulceration. Ten patients had only limb involvement, two had chest wall involvement, one had extension from right leg to the abdominal wall and one had extension from the left buttock to perineum/labia. STSS occurred in nine patients. Ten patients required inotropic support, six had a documented coagulopathy and eleven had renal impairment. Thirteen patients (93%) required ICU stay of 1–24 days. Eleven patients were ventilated, and six received renal replacement therapy.9/L) in only 5/14 (36%) patients, although lymphocyte count was globally reduced, being <1.0 × 109/L in all patients, and <0.5#215;109/l in nine cases (64%). Renal function was frequently impaired, with 11/14 (79%) patients having raised urea (mean 17.7 mmol/L, NR 3.0–8.0 mmol/L) and 12/14 (86%) having raised creatinine (mean 301 μmol/L, NR 50–100 μmol/L). All patients were hypoalbuminemic with 11 having albumin <30 g/L (mean 24.5, range 11–34, NR 35–45 g/L) Plasma creatine kinase (CK) ranged from 25 U/L to 2526 U/L (NR <220 U/L), being elevated in 5/9 (56%) patients measured. C-Reactive Protein (CRP) was substantially raised in all 11 where measured, being between 108–522 mg/L (NR<10). Blood cultures were positive for GAS in seven cases (50%). GAS was isolated from all operative tissue specimens, with four also positive for S. aureus.Total white cell count (WCC) was found to be raised , one being emm sequence type 3.2, and the other three isolates in this cluster were all different emm types.One GAS isolate could not be sequenced. GAS from the other 13 patients showed a large diversity of All patients underwent surgery 0–7 days after presentation. Debridement alone was conducted in nine patients, and combined with fasciotomy in one case. Fasciotomy alone was done in one case. Three patients underwent limb amputation. At surgery it was established that three patients (21%) had concurrent myonecrosis. Antibiotic therapy was instituted with a beta lactam in all patients (benzylpenicillin in nine, meropenem in three, cephazolin in one and flucloxacillin in one). Clindamycin was also given in eleven patients. Adjuvant hyperbaric oxygen therapy was given in four cases, and intravenous immunoglobulin (IVIG) in one.Four patients (29%) died, all being Aboriginal females with co-morbidities. Two of the four patients with truncal infection died in comparison to only two of the ten with only peripheral involvement. The mean time to surgery from presentation was 4.25 days in patients who died and 3.7 days in survivors (not significant). Blood cultures were positive in 3/4 fatalities with STSS in 3/4. All four were in ICU for 1–5 days until death, with all requiring inotropes and mechanical ventilation, two given renal replacement therapy and one IVIG. Two received clindamycin in addition to beta-lactam antibiotic therapy. Surgery in the fatal cases consisted of debridement and/or fasciotomy, with no amputation.We describe a series of 14 cases of GAS necrotising fasciitis in a region with a high background incidence of GAS disease. Our incThe mean age of our patients 41.5 years) is lower than other reports showing an average age of 56–58 years, with notable increases in incidence with increasing age1.5 years,15. 79% 9/L, 64% of those being <0.5 × 109/L. Low albumin was also universal.Two studies have looked at the value of early blood test results in predicting NF as opposed to non-NF soft tissue infection. One found that CK >600 U/L achieved PPV of 58% for NF. HoweverSTSS occurred in 64% of our cases in comparison to 40–46% in other series, and reiCase-fatality rates for NF previously reached 50%, with mortality of 80–100% in GAS myositis. Recent Immediate, extensive surgical debridement of all necrotic tissue is considered essential for optimal treatment of NF, with one early observational study showing a mortality of 0% with aggressive early surgery in comparison to 50% in historical controls,20. SurgCombination therapy with benzylpenicillin and clindamycin is now recommended treatment for proven GAS NF and STSS-23. ExpeHyperbaric oxygen (HBO) therapy has been advocated as adjuvant therapy for both microbiological types of NF. Although there are no randomised trials, NF is an approved Undersea and Hyperbaric Medical Society indication for HBO therapy. It is sThe clinical picture of STSS has been attributed to superantigens produced by GAS, including pyrogenic exotoxins, streptococcal superantigen SSA and a mitogenic exotoxin. This isIn summary, there is in vitro and in vivo experimental data to suggest a benefit for clindamycin, HBO therapy and IVIG in GAS NF and myositis. This is supported by limited case series but not by randomized controlled trials. The primary role of early, aggressive surgery in GAS NF remains probably the most critical factor in minimizing mortality. Clindamycin is recommended for GAS NF but whether clindamycin alone is as effective as penicillin plus clindamycin remains unclear. Benefit from HBO remains unproven, while there is emerging evidence for the use of IVIG, especially if STSS is present.emm type 1. This reflects previous findings of enormous diversity amongst organisms causing GAS bacteremia in our region, with no evidence of a dominant clone[Following a reported resurgence in invasive GAS infections in the 1980's, epidemiological studies proposed that dissemination of a new, more virulent strain of M1 serotype GAS was the cause for a large proportion of these infections. In repoant clone.Emm sequencing typing of GAS isolates showed none to be the same and only 1/13 to be emm1. GAS NF in our region is not clonal but occurs from a wide diversity of GAS strains that reflect the very high background rate of both uncomplicated GAS infection and invasive disease. While NF case clusters can occur from a single emergent GAS clone, this is not evident in our tropical region. The specific virulence factors of the GAS which do cause NF and the basis of the inadequate host response in those patients who develop NF on infection with these GAS require further elucidation.In conclusion we have described a case series of necrotising fasciitis due to GAS for a unique mixed population of patients living in both affluent and disadvantaged society. Our overall incidence rates of GAS NF are higher than previously reported, with rates in the Aboriginal population up to five times those previously published. The majority of those with NF had underlying chronic illnesses and mortality was also higher in this group. Important laboratory findings were that leukocytosis was absent in more than half of cases but all had substantial lymphopenia. The authors declare they have no competing interests.MH collated and analysed the case data and was principal writer of the manuscript, PF carried out the molecular studies, PC participated in the study design and patient management, BC conceived the study and participated in the study design and coordination and patient management. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Studies on the effects of ethylene on CO2 metabolism have shown conflicting results. Increase or inhibition of photosynthesis by ethylene has been reported. To understand the physiological processes responsible for ethylene-mediated changes in photosynthesis, stomatal and mesophyll effects on photosynthesis and ethylene biosynthesis in response to ethephon treatment in mustard (Brassica juncea) cultivars differing in photosynthetic capacity were studied.The stimulatory effect of CON), stomatal conductance (gS), carbonic anhydrase (CA) activity, 1-aminocyclopropane carboxylic acid synthase (ACS) activity and ethylene evolution were similar in both the cultivars. Increasing ethephon concentration up to 1.5 mM increased PN, gS and CA maximally, whereas 3.0 mM ethephon proved inhibitory. ACS activity and ethylene evolution increased with increasing concentrations of ethephon. The corresponding changes in gs and CA activity suggest that the changes in photosynthesis in response to ethephon were triggered by altered stomatal and mesophyll processes. Stomatal conductance changed in parallel with changes in mesophyll photosynthetic properties. In both the cultivars ACS activity and ethylene increased up to 3.0 mM ethephon, but 1.5 mM ethephon caused maximum effects on photosynthetic parameters.The effects of ethephon on photosynthetic rate (PS were a response maintaining stable intercellular CO2 concentration (Ci) under the given treatment in both the cultivars. Also, the high photosynthetic capacity cultivar, Varuna responded less to ethephon than the low photosynthetic capacity cultivar, RH30. The photosynthetic capacity of RH30 increased with the increase in ethylene evolution due to 1.5 mM ethephon application.These results suggest that ethephon affects foliar gas exchange responses. The changes in photosynthesis in response to ethephon were due to stomatal and mesophyll effects. The changes in g N) have been reported. It has been shown to increase PN [N with ethylene-releasing compounds was due to the increase in chlorophyll per unit leaf area [N in response to ethephon treatment. For that, PN, stomatal conductance (gS) and carbonic anhydrase (CA) activity were recorded. To find a possible relationship of ethylene-mediated changes in foliar gas exchange parameters, activity of ACS and ethylene evolution were also determined. The work was carried out in two cultivars of mustard previously shown to have different photosynthetic capacity [Photosynthesis is controlled by several intrinsic and extrinsic factors. Of these, plant hormones have received considerable attention in the past in photosynthetic responses of plants. Ethylene is a phytohormone that influences every aspect of plant growth and development . It is srease PN -7 or decrease PN ,9, but neaf area  or by greaf area . In my eeaf area . The goacapacity .N, gS and CA were found significant in both the cultivars  decreased the characteristics in both the cultivars. The ratio of intercellular to ambient CO2 concentration (Ci/Ca) was constant.The effects of ethephon on P Figures , 2. EtheN, gS and CA. Ethylene evolution with 3.0 mM ethephon proved inhibitory for photosynthetic parameters.Ethephon application significantly affected ACS activity and ethylene evolution, and were greatest with 3.0 mM ethephon Table . Low phoN due to ethephon has been reported [s and CA values in both the cultivars showed stomatal and mesophyll effects on photosynthesis. Mesophyll effects are characterized as a product of CO2 binding capacity and the electron transport capacity. The carboxylation capacity determines the mesophyll effects [2 fixation exhibited the enhanced carboxylation reaction [2 concentration (Ci) under the given treatment. Thus, stomatal and mesophyll processes contributed to the increase in PN in response to ethephon. The ethephon-induced effects on photosynthetic parameters were mediated by ethylene evolved due to ethephon treatment. Taylor and Gunderson [S and ethylene-enhanced PN. Higher concentration of ethephon (3.0 mM) decreased the PN and gS. Such condition of inhibition of PN by ethylene-releasing compound has been observed by Kays and Pallas [N due to its effect on gS. Mattoo and White [2 assimilation and the plant responded depending on the tissue concentration. On the similar lines, Dhawan et al. [et al. [2 regulated PN and was related to ethylene evolution. In the present study, low photosynthetic cultivar, RH30 responded more to ethephon than the high photosynthetic cultivar, Varuna. In control plants, lesser ethylene evolution in RH30 than Varuna was responsible for lesser PN. As the ethylene evolution increased with ethephon application, the capacity of RH30 for PN also increased resulting in higher per cent increase in PN than the Varuna. An increase of 75% ethylene in RH30 due to 1.5 mM ethephon increased PN by 41.8%, whereas 52.6% increase in ethylene in Varuna due to the same treatment increased PN by 31.8%. Earlier strong positive correlation between ACS activity and PN has been shown [Maximum rates of photosynthesis were found with 1.5 mM ethephon. The increase in Preported -6. Incre effects ,14. Incrreaction -17. The underson  showed ad Pallas  and Rajad Pallas . In all nd White  reportedn et al. , Kao andn et al.  and Grod [et al.  reasoneden shown .It therefore, appears possible that the threshold value for ethylene with 1.5 mM ethephon was comparable to that which elicits the ethylene-mediated hormonal responses, which differ with the cultivars inherent capacity of physiological processes. It is that there is some requirement of ethylene for optimum response. Low and high concentration represent the two ends of an optimum curve, promoting at low concentration and inhibiting at high.N in both high and low photosynthetic capacity cultivars, Varuna and RH30. In both the cultivars, changes in PN were due to stomatal and mesophyll effects. Ethephon-induced PN was attributed to ethylene evolution. The high photosynthetic capacity cultivar, Varuna responded less to ethephon than the low photosynthetic capacity cultivar, RH30. The low PN of RH30 was due to low level of ethylene. The low photosynthetic capacity of RH30 could be enhanced to give higher PN through increase in ethylene evolution. However, for both the cultivars there is a range of physiologically active concentration of ethylene beyond which it exerts inhibitory effects.This study shows that ethephon affects PBrassica juncea L. Czern & Coss.), namely Varuna (high photosynthetic capacity) and RH30 (low photosynthetic capacity) were grown from seeds in 10 m2 field plots in complete randomized design with five replications. At seedling establishment a plant population of 12 plants m-2 was maintained and recommended plant cultivation procedures were adopted. A uniform recommended soil application of 18 g N, 3 g P and 3 g K m-2 was given at the time of sowing so as the nutrients were non-limiting.Two cultivars of mustard (At 30 d after sowing, 0, 0.75, 1.5 and 3.0 mM ethephon (2-chloroethyl phosphonic acid) was sprayed with a hand sprayer. Ethephon is a direct ethylene source when applied to plants and elicits response identical to those induced by ethylene gas ,24. SincN, gS, Ci, CA activity, ACS activity and ethylene evolution were determined.At 45 d after sowing (15 d after ethephon treatment) PN, gS and Ci were measured using infrared gas analyzer (LiCOR 6200, Lincoln, NE) on fully expanded upper most leaves at saturating light intensity on four plants from each replicate. The atmospheric conditions during the experiment between 1100–1200 h were: photosynthetic active radiation about 1050 μmol m-2 s-1, relative humidity 64% and temperature 23°C, atmospheric CO2 concentration 360 μmol mol-1.P2 during the catalytic action of enzyme on sodium bicarbonate was estimated by titrating the reaction mixture against 0.05 N hydrochloric acid.The leaves used for photosynthesis measurement were selected for CA activity determination. CA was measured by the method of Dwivedi and Randhava . Leaves et al. [et al. [2 followed by 0.1 mL of 1:1 mixture of saturated NaOH/NaCl and incubated on ice for 10 min, and ethylene evolution was measured on a gas chromatograph. For control set AdoMet was not added. For ethylene evolution 5 mL of gas phase was removed with a syringe and ethylene was measured on a gas chromatograph (GC 5700, Nucon, New Delhi) equipped with 1.8 m Porapack N (80/100 mesh) column, a flame ionization detector and an integrator. Nitrogen was used as carrier gas. The flow rates of nitrogen, hydrogen and oxygen were 0.5, 0.5 and 5 mL s-1, respectively. The oven temperature was 100°C and detector was at 150°C. Ethylene identification was based on the retention time and quantified comparing with the peaks from standard ethylene concentrations.Activity of ACS was measured adopting the methods of Avni et al.  and Woes [et al. . Leaf tiData were analyzed statistically and standard error of the mean value was calculated. Analysis of variance was performed to identify the significant differences among treatments at P < 0.05 .i – intercellular CO2 concentration; gS – stomatal conductance; PN – net photosynthetic rateAdomet – S-adenosyl methionine; ACS – 1-aminocyclopropane carboxylic acid synthase; CA – carbonic anhydrase; C


For two centuries, vaccination has been the dominating approach to develop prophylaxis against viral infections through immunological prevention. However, vaccines are not always possible to make, are ineffective for many viral infections, and also carry certain risk for a small, yet significant portion of the population. In the recent years, FDA's approval and subsequent market acceptance of Synagis, a monoclonal antibody indicated for prevention and treatment of respiratory syncytial virus (RSV) has heralded a new era for viral infection prevention and treatment. This emerging paradigm, herein designated "Biochemical Prevention and Treatment", currently involves two aspects: (1) preventing viral entry via passive transfer of specific protein-based anti-viral molecules or host cell receptor blockers; (2) inhibiting viral amplification by targeting the viral mRNA with anti-sense DNA, ribozyme, or RNA interference (RNAi). This article summarizes the current status of this field. A landmark in the battle against viral infectious diseases was made in 1798 when Jenner first inoculated humans against smallpox with the less virulent cowpox. For about two centuries since then, humans relied almost exclusively on vaccines for protection against viruses. Only in the recent years, new strategies for controlling viral infectious diseases have emerged, which have so far led to a couple of viral prophylaxis/therapeutics on the market. These strategies are fundamentally different from vaccines in that they attempt to directly interrupt viral infectious life cycle at molecular level by using proteins or oligonucleotides. To differentiate them from the conventional vaccines that prevent viral infection by boosting immune system, we refer the new antiviral approaches as "Biochemical Prevention and Treatment" . Soluble receptor drug candidates have gradually lost favor over the past several years due to issues relating to low potency and cost. Peptide-based drug candidates are limited by insufficient efficacy and unfavorable pharmacokinetics. MAbs have increasingly gained favor in large part because of the development of chimeric, humanized, and human antibodies have reduced the immunogenicity of antibody therapies. The MAbs that are currently in clinical trials for viral infection prophylaxis and treatment are listed in Table The respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in infants and young children producing bronchiolitis and pneumonia worldwide. RSV infection leads to more than 90,000 hospitalizations and a 2% mortality rate among infants nationwide . ApproxiThe most successful approach to date has been Biochemical Prevention and Treatment with anti-viral antibodies. In 1996, RespiGam™  became available for use in children less than two years of age with high-risk factors -10. The in vitro studies have reported effective viral inhibition by receptor-blocking MAbs. However, these works have not yielded yet any approved drug on the market.Human rhinovirus (HRV) causes over 80% of the common cold in the fall . Developin vitro [In HRV infection, about 90% of HRV serotypes utilize a single cell surface receptor exclusively, which is the intercellular adhesion molecule-1 (ICAM-1), for viral attachment and subsequent viral entry ,18. As sin vitro -21. Admiin vitro -24. For in vitro .in vitro assays, CFY196 consistently and significantly outpaced the best commercial anti-ICAM-1 MAbs in preventing HRV infection as measured by reduction of cytopathic effects and HRV viral titers [High avidity is achieved by multivalency. To improve avidity of HRV receptor blocking antibody, a novel tetravalent recombinant antibody, CFY196, has been generated against ICAM-1 . CFY196 l titers . The prel titers . Further®. A number of clinical trials of drug candidates from these technologies are currently ongoing.Targeting viral mRNA is one of the most active areas of research and development. Several strategies have emerged over the years and are being tested pre-clinically and clinically. They include: antisense-oligonucleotides (AS-ONs), ribozymes, and recently, RNA interference (RNAi). All these strategies share the features of conceptual simplicity, straightforward drug design and quick route to identify drug leads. However, the challenges have been to improve potency, pharmacokinetics and, most importantly, intracellular delivery of the drug candidates. As the oldest strategy, AS-ON technology has produced to date one drug in the market place, VitraveneAntisense-oligonucleotides (AS-ONs) are short synthetic oligonucleotides that form complementary pair with specific viral mRNA targets. AS-ONs inhibit viral protein production by both blocking viral mRNA translation and triggering its degradation. Since the discovery of viral inhibition effect of AS-ONs by Zamecnik and Stephenson in 1978 , antisenVitravene is the first AS-ON based drug approved by FDA. Vitravene, or fomivirsen sodium, is a 21-base phosphorothioate oligodeoxynucleotide complementary to the messenger RNA of the major immediate-early region proteins of human cytomegalovirus, and is a potent and selective antiviral agent for cytomegalovirus retinitis, a herpes-like eye disease that afflicts the immune-suppressed ,30. A nufirst generation' DNA analogs. The 'second generation' ONs contain nucleotides with alkyl modifications at the 2' position of the ribose. They are less toxic than PS-DNAs and have a slightly enhanced affinity. DNA and RNA analogs with modified phosphate linkages, or different sugar residues substituting the furanose ring have been referred as 'third generation' [Phosphorothioate (PS) oligodeoxynucleotides are the 'eration' . For insRibozymes (Rz) are catalytically active ONs that both bind and cleave target RNAs. They were discovered after the AS-ON technology. Initial findings on ribozymes raised the hope that they may offer a more potent alternative to AS-ONs. Many cell based and animal tests have performed on anti-viral effects of ribozymes, including HIV, hepatitis B, hepatitis C, influenza, etc. Results from these tests have shown that ribozymes are promising viral inhibitors -38. Howein vitro synthesized small interfering RNAs (siRNAs) of 21 to 23 nucleotides in length can effectively silence targeted genes in mammalian cells without triggering interferon production [RNA interference, or RNAi, is the inhibition of expression of specific genes by double-stranded RNAs (dsRNAs). It is becoming the method of choice to knockdown gene expression rapidly and robustly in mammalian cells. Comparing to the traditional antisense method, RNAi technology has the advantage of significantly enhanced potency; therefore, only lower concentrations may be needed to achieve same level of gene knockdown. RNAi gained rapid acceptance by researchers after Tuschl and coworkers discovered that oduction ,41. In moduction .Several initial studies, which test the potential application of synthetic siRNAs as antiviral agents, have shown very promising results. To date, RNAi has been used effectively to inhibit the replication of several different pathogenic viruses in culture, including: RSV  , influenCurrently, our understanding of the biological mechanisms underlying RNAi lags behind the movement to apply this technology to human diseases such as viral infections. Some major technical hurdles need to be overcome before siRNA-based anti-viral prophylaxis and treatments move into the clinics. Especially, intracellular delivery of siRNA needs to be greatly improved. The next few years of research will indicate whether RNAi technology will realize its potential as the next wave of Biochemical Prevention and Treatment.Dr. Hervé Le Calvez declares that he has no competing interest. Dr. Mang Yu and Dr. Fang Fang are the co-founders and current share holders of Perlan Therapeutics who has developed CFY196.


Molecular Cancer offers Open Access to all of its content, thereby providing a platform to present information to specialists and the public in order to further promote free exchange of ideas, concepts and findings in all fields of cancer-related biomedical science. All the published articles in the journal are determined by the peer review process.Open Access is one of the attempts to maximize the exchange of information, and therefore benefits the scholar communication . MoleculOpen Access has following broad benefits for science and the general public:• All articles become freely and universally accessible online; so an author's work can be read by anyone at no cost.• The authors hold copyright for their work and grant anyone the right to reproduce and disseminate the article, provided that it is correctly cited.• A copy of the full text of each Open Access article is permanently archived in an online repository separate from the journal, such as PubMed Central, the US National Library of Medicine's full-text repository of life science literature, the repositories at the University of Potsdam in Germany, at INIST in France and in e-Depot, the National Library of the Netherlands' digital archive of all electronic publications.• Authors are assured that their work is disseminated to the widest possible audience. This is accentuated by the authors being free to reproduce and distribute their work, for example by placing it on their institution's website. It has been suggested that free online articles are more highly cited because of their easier availability .• The information available to researchers will not be limited by their library's budget, and the widespread availability of articles will enhance literature searching.• The results of publicly funded research will be accessible to all interested readers and not just those with access to a library with a subscription. As such, Open Access could help to increase public interest in, and support of, research. Please note that this public accessibility may become a legal requirement in the USA if the proposed Public Access to Science Act is made law .• A country's economy will not influence its scientists' ability to access articles because resource-poor countries (and institutions) will be able to read the same material as wealthier ones, although creating access to the internet is another matter.Molecular Cancer published a number of interesting papers, and the list of the top ten most accessed articles is available at . All papers accepted by Molecular Cancer appear as 'accepted manuscript' on the web pages and are subsequently included in PubMed. A fully formatted portable document file is available approximately two to three weeks after acceptance along with a web-version of the article.The on-line publication, to the exclusion of print, has many advantages: Coloured pictures can be presented along with large sets of supporting data (movies, tables, pictures, et cetera) without additional charges. In addition, the on-line submission process allows a fast and effective handling of papers and allows authors to check the status of their submitted manuscript(s). There is no limitation in space, but concise papers are more likely to be read.Molecular Cancer, all members of the editorial board and the reviewers for their ongoing support for Open Access publishing and for aiming higher standards for Molecular Cancer.The peer review policy, described in , ensuresMolecular Cancer did not change significantly, compared to the last report [Molecular Cancer after revisions. In addition to indexing in PubMed, PubMed Central and other search engines, Molecular Cancer is working closely with the Institute for Scientific Information to ensure that citation analysis of our articles will be available.The acceptance rate of t report . One outPJC is Editor-in-Chief and CS is Deputy Editor of this journal. Both do not receive any remuneration for their efforts but they are exempted from the article processing fee for this journal.


Propagation of repolarization is a phenomenon that occurs in cardiac muscle. We wanted to test whether this phenomenon would also occur in our model of simulated action potentials (APs) of cardiac muscle (CM) and smooth muscle (SM) generated with the PSpice program.gj) across the four cell junctions.A linear chain of 5 cells was used, with intracellular stimulation of cell #1 for the antegrade propagation and of cell #5 for the retrograde propagation. The hyperpolarizing stimulus parameters applied for termination of the AP in cell #5 were varied over a wide range in order to generate strength / duration (S/D) curves. Because it was not possible to insert a second "black box" (voltage-controlled current source) into the basic units representing segments of excitable membrane that would allow the cells to respond to small hyperpolarizing voltages, gap-junction (g.j.) channels had to be inserted between the cells, represented by inserting a resistor (Rm) to within the range of the sigmoidal curve of the Na+ conductance (CM) or Ca++ conductance (SM) terminated the AP in cell #5 in an all-or-none fashion. If there were no g.j. channels (Rgj = ∞), then only cell #5 repolarized to its stable resting potential . The positive junctional cleft potential (VJC) produced only a small hyperpolarization of cell #4. However, if many g.j. channels were inserted, more hyperpolarizing current was required (for a constant duration) to repolarize cell #5, but repolarization then propagated into cells 4, 3, 2, and 1. When duration of the pulses was varied, a typical S/D curve, characteristic of excitable membranes, was produced. The chronaxie measured from the S/D curve was about 1.0 ms, similar to that obtained for muscle membranes.Application of sufficient hyperpolarizing current to cell #5 to bring its membrane potential (VJC) developed in the narrow junctional clefts. Because it was not possible to insert a second black-box into the basic units that would allow the cells to respond to small hyperpolarizing voltages, only cell #5 (the cell injected with hyperpolarizing pulses) repolarized in an all-or-none manner. But addition of many g.j. channels allowed repolarization to propagate in a retrograde direction over all 5 cells.These experiments demonstrate that normal antegrade propagation of excitation can occur in the complete absence of g.j. channels, and therefore no low-resistance pathways between cells, by the electric field (negative V In c muscle ,14,15, aJC) was large and sufficient to allow transfer of excitation to the contiguous cell, without the requirement of gap-junction (g.j.) channels. Propagation of excitation can occur by the EF mechanism alone, even when the excitability of the cells was made low. In connexin-43 (heterozygous) and Cx40 knockout mice, propagation in the heart still occurs, but it is slowed [We recently modeled propagation of APs of cardiac muscle and smooth muscle using the PSpice program for circuit design and analysis -18. Likes slowed -22 as prThe present experiments were carried out to study propagated repolarization in this model of simulated action potentials (APs). Propagation of repolarization is a phenomenon that occurs in cardiac muscle . It has JC) was placed in the junctions between adjoining cells. The basic units were connected internally by the intracellular longitudinal resistance (ri). The basic units were connected externally with the extracellular resistance (RO), broken down into a longitudinal component (Rol) and a transverse  component (Ror). RO was connected to ground as depicted in Figure The methods used and PSpice program (Cadence Co, Portland) have been described in detail previously, including the circuit ,18. In b+ compared to Na+ (cardiac muscle) or Ca++ (smooth muscle) were set to give resting potentials (RPs) of -80 mV for cardiac muscle and -55 mV for smooth muscle. During excitation, the action potentials (APs) overshot to +32 mV and +11 mV, respectively.The myocardial cell was assumed to be a cylinder 150 μm long and 16 μm in diameter, and the smooth muscle cell a cylinder 200 μm long and 5 μm diameter. Since in vascular smooth muscle (VSM), the muscle fibers run in a circular direction, if transverse velocity is calculated, the fiber diameter should be used. The values of the capacitive and the resistive elements in each basic unit were set to reflect the input resistance (ca 20 MΩ) and input capacitance (ca 100 pF) of the individual cells, and the junctional units were prorated, with respect to the surface units, based on relative areas represented. At rest, the resistance of KS1) were always applied internally to the first cell of the chain (cell A1). Rectangular depolarizing current pulses of 0.25 nA amplitude and 0.50 ms duration were applied. The delay time before the IS1 pulse was applied was usually set to 1.0 ms in SM. A second stimulus (IS2) that was hyperpolarizing was applied to the inside of the last cell (A5) of the chain when the APs of all 5 cells were in their plateau phase. The intensity and duration of the IS2 pulses were varied over a wide range in order to generate strength / duration (S/D) curves.Electrical stimulations (I+ or Ca++ conductance during excitation), this function had to be done with a V-controlled current source (our "black-box"). The sigmoidal relationship between conductance and membrane potential (VM), over a relatively narrow VM range, was mimicked by the black-box. The Na+ or Ca++ current required for excitation had to be calculated for several VM values and inserted into the GTABLE function.Because the PSpice program does not have a voltage-dependent resistance (to generate the increase in Nagj) across each cell-to-cell junction. This resistor connected the inner surface of the prejunctional membrane with the inner surface of the postjunctional membrane. This Rgj shunt resistance was varied between 10,000 MΩ (1 tunnel), 1000 MΩ , 100 MΩ (100 tunnels), 10 MΩ (1,000 tunnels), and 1.0 MΩ (10,000 tunnels). Each tunnel was assumed to have a conductance of 100 pS.Experiments were done with a single chain of 5 cells or 2 cells. There were no gap junctions between the cells of the chain under initial conditions. The presence of gap junction connexons (tunnels) was represented by adding a variable shunt resistance (RS1) produced propagation of APs down the chain. At the plateau (peak) of the APs, a repolarizing pulse applied intracelluarly to cell A5, if of sufficient intensity (duration constant), produced a sudden repolarization of only cell A5  repolarization of the stimulated cell (A5) of the 5-cell chain in both cardiac muscle Fig.  and smoocal case -25.+ leg of the basic circuit, because the PSpice program became erratic. Therefore, in order to achieve propagation of the repolarization of cell A5 in the retrograde direction, it was necessary to insert gap-junction channels between the cells of the chain (1, 10, 100, 1000, 10000 channels). This corresponded to adding resistive shunts between the cells across the junctions (Rgj) of 10000, 1000, 100, 10, and 1.0 MΩ (assuming each channel has a conductance of 100 pS).As indicated in the Methods section, it was not possible to insert a second black-box in the KThe results of doing such an experiment are shown in Fig S2) applied to cell A5 were varied over a wide range in order to generate strength / duration curves. This was done when Rgj was infinite (i.e., 0 channels) and when Rgj was 10 MΩ (1000 channels) for strong coupling. The pulse duration was initially constant at 1.0 ms  and then lowered to 0.5 ms and to 0.25 ms. The current intensity was varied until the sharp endpoint occurred, namely the stable repolarization of all cells in the chain. These results are plotted in Figure Panel A is the strength / duration (S / D) curve for when Rgj was infinite (0 channels), and Panel B is the S/D curve for when Rgj was 10 MΩ(1000 channels). Note that the IS2 intensity was about 8–10-fold greater when the cells were well-coupled, because the applied hyperpolarizing current had to spread to all 5 cells of the chain. Regardless, the chronaxie values were about the same (ca. 1.0 ms).The intensity (strength) and duration of the rectangular hyperpolarizing current pulses  injected with repolarizing current (IS2) was able to repolarize in an all-or-none manner. The neighboring cell (A4) exhibited only a slight repolarization of <1 mV when cell A5 had repolarized completely back to the RP (-80 mV for CM and -55 mV for SM). This fact emphasized that there were no low-resistance connections between the cells under our initial conditions.In principle, the addition of a second black-box into the Kgj values of 1.0, 10, and 100 MΩ) did allow propagation of repolarization to occur. The borderline value was 10 g.j. channels (1000 MΩ Rgj), e.g., repolarization propagated part-way down the chain in SM and almost succeeded in CM. Of course, inserting the g.j. channels required that the IS2 repolarizing current applied be much greater. This is because the IS2 current had to spread down the entire chain, with the threshold current required to cause all cells to repolarize being determined by sufficient current entering distal cell A1 to repolarize it to the GTABLE sigmoidal region. Thus the proximal cells, like A5 and A4, became hyperpolarized beyond the level required for their repolarization.However, addition of 10,000, 1,000, or 100 g.j channels  and when all 5 cells repolarized (Rgj of 1.0, 10, and 100 MΩ). Plots of current intensity (ordinate) versus current duration (abscissa) gave the typical hyperbolic strength/ duration curve for excitable membranes. The chronaxie values were about 1.0 ms, for which a time constant τm of about 1.44 ms could be calculated. The S/D curves for the two conditions (Rgj = ∞ and Rgj = 10 MΩ) show that the current intensity required was about 8–10-fold greater when there were many gj-channels, in both CM and SM.The repolarizing Ir) varied with the number of gj-channels (Table r was about 200 cm/s in the very well coupled case (10,000 channels) and about 50 cm/s in the less coupled case (100 channels) (Table r) was much lower than the velocity for antegrade propagation (θa.). In the 2-cell chain, the calculated velocities of propagated repolarization were similar to those for the 5-cell chain . Third, it shows for the first time that, in the PSpice model, the membranes exhibit the characteristic strength/duration curves. Fourth, it shows that the PSpice program has some serious limitations.The present study provides some new and important information about the PSpice simulations. First, it verifies that propagation (orthodromic) can occur in the complete absence of gap-junction channels, as previously reported ,4,16-18.S2 current was able to repolarize. Since the potential change in the neighboring cell was only about 1 mV or less, this emphasizes that there were no low-resistance connections between the simulated cells under initial conditions. Propagation in the orthodromic direction occurs by the electric field (EF) discussed in previous papers (1–4, 16–18). The repolarizing IS2 current gave S/D plots that were typical rectangular hyperbolic curves for excitable membranes, with chronaxie values of about 1.0 ms, both for CM and SM. The calculated velocity for propagated repolarization was greater when the number of gj-channels was increased. The antidromic (retrograde) propagation velocity was usually considerably slower than the orthodromic (antegrade) propagation velocity for depolarization. The present findings do not necessarily imply that, in biological tissue, gap junctions are required for propagated repolarization to occur.In summary, because of technical difficulties with the PSpice program, it was necessary to insert gj-channels in order to produce propagation of repolarization. Otherwise, only the modeled cell injected (A5) with the repolarizing I


Seroma formation is the most frequent postoperative complication after breast cancer surgery. We carried out a study to investigate the effect of various demographic, clinical and therapeutic variables on seroma formation.A retrospective cross sectional study of patients who underwent surgical therapy for breast cancer with either modified radical mastectomy (MRM) or breast preservation (BP) was carried out. The demographic data and clinical information were extracted from case records. Seroma formation was studied in relation to age, type of surgery, tumor size, nodal involvement, preoperative chemotherapy, surgical instrument , use of pressure garment, and duration of drainage. The multiple logistic regression analysis was performed to estimate odds ratios.A total of 158 patients with breast cancer were studied. The mean age of the patients was 46.3 years (SD ± 11.9). Seventy-three percent underwent modified radical mastectomy and the remaining 27% received breast preservation surgery. Seroma occurred in 35% of patients. In multivariate logistic regression analysis an association of postoperative seroma formation was noted with modified radical mastectomy (OR = 2.83, 95% CI 1.01–7.90, P = 0.04). No other factor studied was found to significantly effect the seroma formation after breast cancer surgery.The findings suggest that the type of surgery is a predicting factor for seroma formation in breast cancer patients. Breast cancer is the second leading cause of cancer death among women. The surgical treatment of choice for these patients is either modified radical mastectomy or breast preservation depending upon stage of the disease. Seroma formation is the most frequent postoperative complication after breast cancer surgery. It occurs in most patients after mastectomy and is now increasingly being considered side effect of surgery rather than a complication however, all patients are not clinically symptomatic . Seroma A cross sectional study of a consecutive sample of 158 patients attending the breast cancer clinic between January 2000 to October 2002 in Tehran, Iran, was carried out. All patients undergoing surgical therapy [modified radical mastectomy (MRM) or breast preservation (BP)] were included. Level II axillary lymph node dissection was performed for both groups. None of the patients underwent immediate reconstruction. The demographic data and clinical information were extracted from case records. Axillary seroma was defined as any clinically apparent fluid collection in the axilla or under the skin flaps and was treated with multiple needle aspirations. Seroma formation was studied in relation to age, type of surgery, tumor size, nodal involvement, preoperative chemotherapy, surgical instrument , use of pressure garment, and duration of drainage. To analyze data univariate odds ratio (or relative risk) was calculated using Chi-square tests or regression analysis and this was followed by the multivariate logistic regression analysis to evaluate independent risk factors related to seroma formation. The variables of interest were selected in a single step (enter method), classification cut off was  set at 0.5, probably of step for entry into the model was set at 0.05 and  removal at 0.1, and the model was set to converge in maximum of 20  iterations. All variables under study were considered as independent predicting factors and seroma formation was considered as dependent variable for multivariate analysis. The study was approved by the institutional ethics committee.2 = 4.52, df = 1, P = 0.03) indicating that those who underwent MRM had higher rate of positive axillary nodes compared to those who received BP (78% vs. 21% respectively). Thirty-one mastectomies were performed by scalpel dissection of the skin flap (20%) and 127 by cautery dissection (80%). Two closed suction drains were placed in all patients undergoing surgery. Sixty-six percent of patients (n = 104) were node positive and the remaining 34% (n = 54) were node negative. The patients' characteristics and univariate odds ratios are shown in Table In all, 158 breast cancer patients were recruited into the study and 55 patients developed seroma, giving an overall incidence of 35% for seroma formation after breast surgery. The mean age of patients was 46.3 years (SD ± 11.9). One hundred and fifteen patients (73%) underwent MRM and BP was performed in 43 patients (27%). The axillary node involvement was significantly different between MRM and BP patients . Of patients with BP, 10 of 43 (23%) developed seroma, while those who underwent MRM 45 of 115 (39%) developed seroma. The seroma formation did not show any significant association with any other variables studied. The results of maultivariate analysis are shown in Table Breast cancer is the most common malignancy in women. Surgery is the mainstay of treatment. Modified radical mastectomy with or without reconstruction or breast preservation in addition to axillary lymph node dissection are common surgical procedures in breast cancer. Surgery of the axilla is associated with numerous complications, including infection, lymphedema of the ipsilateral upper extremity and collection of fluid in surgical site (seroma). Most common complication after breast cancer surgery is wound seroma. The exact etiology of seroma formation remains controversial. Several interventions have been reported with the aim of reducing seroma formation including the use of ultrasound scissors in performing lymphadenectomy , buttreset al reported that the use of electrocautery to create skin flaps in mastectomy reduces blood lose but increased the rate of seroma formation [Several studies have been performed to investigate factors related to post-surgical seroma. These studies have observed that the early removal of drains might led to increased incidence of seroma , whereasormation . In addiormation . Compresormation . Howeverormation  and evenormation .et al, demonstrated that patients who underwent modified radical mastectomy had a greater incidence of seroma formation than patients who underwent breast preservation surgery [et al indicated that the tumor size and total amount of drainage represented the principal factors of seroma formation following axillary dissection in patients underwent surgery for breast cancer [In the present study no relationship was observed between the incidence of seroma and the patients' age, tumor size, and lymph node status. However, the study found that the MRM was associated with higher rate of postoperative seroma formation (OR = 2.83, P = 0.04). Similarly, Gonzalez  surgery . They al surgery . A recent cancer .The results of our study suggest that seroma formation after breast cancer surgery is independent of duration of drainage, compression dressing and other known prognostic factors in breast cancer patients except the type of surgery, i.e there is a 2.5 times higher risk of seroma formation in patients undergoing MRM compared to BP. The small sample size of present study is a limitation and hence the power of the study is low. A number of questions remain unanswered and more research is needed to answer these.The authors declare that they have no competing interests.NoneEH designed the study, collected the data and wrote the first draft of the manuscript.AK, MN, ME and HH all contributed to patient recruitment and the preparation of first draft of the manuscript.AM contributed to the study design, data analysis, and edited the final version.All authors read and approved of the manuscript.


The contribution of low-penetrant susceptibility variants to cancer is not clear. With the aim of searching for genetic factors that contribute to cancer at one or more sites in the body, we have analyzed familial aggregation of cancer in extended families based on all cancer cases diagnosed in Iceland over almost half a century.We have estimated risk ratios (RRs) of cancer for first- and up to fifth-degree relatives both within and between all types of cancers diagnosed in Iceland from 1955 to 2002 by linking patient information from the Icelandic Cancer Registry to an extensive genealogical database, containing all living Icelanders and most of their ancestors since the settlement of Iceland.We evaluated the significance of the familial clustering for each relationship separately, all relationships combined (first- to fifth-degree relatives) and for close (first- and second-degree) and distant (third- to fifth-degree) relatives. Most cancer sites demonstrate a significantly increased RR for the same cancer, beyond the nuclear family. Significantly increased familial clustering between different cancer sites is also documented in both close and distant relatives. Some of these associations have been suggested previously but others not.We conclude that genetic factors are involved in the etiology of many cancers and that these factors are in some cases shared by different cancer sites. However, a significantly increased RR conferred upon mates of patients with cancer at some sites indicates that shared environment or nonrandom mating for certain risk factors also play a role in the familial clustering of cancer. Our results indicate that cancer is a complex, often non-site-specific disease for which increased risk extends beyond the nuclear family. It's not often that an entire nation's genealogy and cancer records are available. But they are in Iceland, and have been used to determine how often cancers occur in families BRCA1 and BRCA2 genes account for around 2%–3% of all breast cancer cases [Highly penetrant susceptibility variants explain only a small fraction of the genetics of all cancer cases. As an example, mutations in the er cases ,5,6,7. HFamily studies have given insight into the contribution of genetic and environmental factors to the etiology of cancer. Case-control, registry- and population-based studies have evaluated familial clustering using either risk ratio (RR) estimations for relatives of cancer patients, or kinship coefficient (KC) estimations for cancer patients. The largest of these studies, utilizing either the Utah Population and Cancer Registry Database or the Swedish Family-Cancer Database, have demonstrated excess familial clustering at practically all cancer sites in the body ,10,11,12Twin studies have also evaluated the role of genes versus environment in cancer susceptibility. The largest study involved close to 45,000 twins from Denmark, Sweden, and Finland where the RR of same type of cancer was calculated for individuals with affected twins and compared to those without an affected twin . The autIn addition to well documented familial clustering for the majority of individual cancers, aggregation of different types of cancers in families has also been observed. Reports have been published on the results of systematic analysis of the aggregation of different cancers using the Utah Population and Cancer Registry Database ,25. In aWe have studied a registry of all cancer cases diagnosed in Iceland from 1 January 1955 to 31 December 2002, with the aim of searching for evidence of genetic factors both at individual cancer sites and those shared by different sites. By cross-referencing cancer prevalence in relatives of cases with the aid of a comprehensive nationwide genealogy database, we have estimated RR separately for first- to fifth-degree relatives of all cancer patients diagnosed in Iceland over 48 y. We demonstrate here an increased cancer risk in relatives outside the nuclear family (third- to fifth-degree relatives) for many cancer sites. These relatives share significant genetic makeup but are less likely to share environmental factors beyond those shared by the general population, indicating that genetic factors may be involved. By applying the analysis across different cancer sites we also demonstrate shared familiality between certain cancer sites both in close and distant relatives. These results suggest that cancer can be considered a broad phenotype with shared genetic factors crossing different cancer sites. That is, the difference between cancers at various sites may in part be the consequence of variable expressivity of the same cancer-predisposing genes.This study was approved by the National Bioethics Committee of Iceland, the Data Protection Authority of Iceland, and the Icelandic Cancer Society. All names of patients listed in the Icelandic Cancer Registry (ICR) and the genealogic database were encrypted through a process approved by the National Bioethics Committee and the Data Protection Authority before being analyzed .The ICR of the Icelandic Cancer Society is a carefully constructed database containing practically complete records of all cancer cases diagnosed in Iceland after 1 January 1955 . RecordsdeCODE Genetics has built a computerized genealogy database of more than 687,500 individuals ,30. The Mates are defined as individuals of the opposite sex who have one or more children in common, regardless of marital status.The RR for relatives is a measure of the risk of disease for a relative of an affected person compared to the risk in the population as a whole. More precisely, for a given relationship the RR for disease B in the relatives of probands with disease A is defined asAP denotes the event that the proband is affected with disease A, and BR denotes the event that the relative is affected with disease B. Note that disease A and disease B can be the same in this definition which applies when estimating RR at individual cancer sites. Using Bayes' rule it can be shown that for symmetric relationships, RR is the same if the roles of A and B are switched, i.e., the RR for disease A in the relatives of probands with disease B is the same as the described above. In this study we always chose the less common phenotype as the proband when estimating RR.where RBO and PAO are the events that the relative and proband are observed with diseases A and B, respectively):A basic underlying assumption in our estimation of RR is that of conditional independence of ascertainment, or censoring, , let n be the size of the population, and finally let x be the number of affected individuals in the population. If P(BR) and P(B | PAR) can reasonably be assumed to be constant in the population, then x/n and a/r, respectively, are estimates of these probabilities. Given these estimates, RR is consistently estimated byThe approach chosen here is to estimate the RR for a number of subpopulations, where prevalence is reasonably constant, and combine them into a single estimate of RR for the full population. Let probands , let a bN subpopulations, such that within each subpopulation P(BR) and P(B | PAR) can be assumed to be constant, although they may vary between subpopulations, and assuming furthermore that RR is the same in all the subpopulations, then the RR is consistently estimated by a convex combination of the estimates for the subpopulations. We selected weights for the combination such that the efficiency of the estimator was at maximum for RR equal to one. Making the simplifying assumption that the relatives are independent , the optimal weight for group j isAssuming the population can be split into j), where a, r, x, and n are defined as above, restricted to the subpopulation j. Note that probands are not restricted to the subpopulation. Given these weights, our estimate of RR is(this is the inverse of the variance of the estimate for RR in subpopulation P(BR) and P(B | PAR) stem from time-dependent censoring of affected status and sex-specific differences. Hence, we have stratified the population so that j runs over groups of people of the same sex and born in the same 5-y periods. For a fixed year-of-birth stratum, there is censoring of affected status (missing data) based on year of onset because of the fact that records cover only the period 1955–2002. Our approach is designed to address this type of missing data. As an example of the stratification, the breast cancer patients in our analysis were born in the years 1865 to 1970 (5-y strata), yielding 35 subpopulations, 22 for female patients, but only 13 for male, as this cancer is rare for males.In this study, the most relevant variations in p values can be calculated using the control groups; thus, a p value of 0.05 for the RR would indicate that 5% of the matched control groups had values as large as or larger than that for the patient's relatives or mates. The number of control groups required to obtain a fixed accuracy of the empirical p values is inversely proportional to the p value. We therefore selected the number of control groups generated adaptively up to a maximum of 100,000. When none of the values computed for the maximum number of control groups were larger than the observed value for the patient's relatives and mates, we report the p value as being less than 0.00001. Using a variance-stabilizing square-root transform, an approximate confidence interval may be constructed based on the distribution of RR for control groups [To assess the significance of the RR obtained for a given group of patients, we compared their observed values with the RR computed for up to 100,000 independently drawn and matched groups of control individuals. Each patient was matched to a single control individual in each control group. The control individuals were drawn at random from the genealogic database with the conditions that they had the same year of birth, the same sex, and the same number of ancestors recorded in the database at five generations back as the matched patients. Empirical l groups .dRR is the RR for relatives of degree d, then dRR – 1 is known to decrease proportional to 2-d as d increases for a monogenetic single variant or additive disease models, and faster for more complex disease models [dRR denoted by As another test for significance of RR between cancer sites, we used combined estimators for risk in relatives of degree 1 and 2 together, degrees 3, 4, and 5 together, and degrees 1 through 5 together. If e models . With thd summed over the relevant degrees. For dRR close to one, the variance of the estimate d for the proband. Based on the Icelandic genealogy for the cancers being studied here, the number of relatives is proportional to γd, where the value of γ quantifies how the number of relatives grows with each degree of relatedness to the proband. This factor γ varies only slightly between cancers and is on average 2.46. Minimizing the variance of the test statistic in equation 6 with respect to the weights yields the statisticwith As above, the choice of weights and the form of the statistic affects only power, not validity. To assess significance, the observed value of the statistic was compared to its value for multiple matched control groups as described above.Although our evaluations of familial clustering, for both close and distant relatives, are based on RR, an alternative approach based on comparing KCs among patients and among controls exists ,24,25. TWe have studied the familial clustering of cancer by estimating RR for first- and up to fifth-degree relatives both within and between all cancer sites. Here we present results for 27 sites that contain 200 or more cancer cases each, based on International Classification of Disease version 10 codes. These 27 sites represent 89% of all cancer cases in the ICR.A significantly increased RR to first-degree relatives of patients with cancer was seen for 22 of the 27 cancer sites . Among tThe medians of the estimated RR values for the 27 sites in first- to fifth-degree relatives were 2.00, 1.32, 1.21, 1.10, and 1.04, respectively.p-values incorporating the increased risk for first- to fifth-degree relatives identified 21 sites being significant at a nominal level of 0.05. Sixteen of those sites remained significant after Bonferroni adjustment for the 27 individual tests   . To discher hand . FourteeThe RR for developing cancer at the same site was also estimated for mates of cancer patients at 22 out of the 27 individual sites. The remaining five sites are sex-specific and calculations thus not applicable. For seven rare cancer sites, affected mates were not observed, corresponding to a RR of zero. Only lung, stomach, and colon cancer were characterized by significantly increased RR values in mates .p values were calculated for all (first- to fifth-degree), close (first- and second-degree), and distant (third- to fifth-degree) relationships. p value, significant at a level of 1 × 10-4, for first- to fifth-degree relationships. This level was significant at the 0.05 level after Bonferroni adjustment for the 351 tests (number of unique pairs of cancers). The strength of the distant familiality  between these pairs of cancers is represented by the thickness of the lines joining sites in We calculated RR between all cancer sites for first- and up to fifth-degree relatives and mates  for distant relationships, of which nine were significant at the 0.001 level. In the latter group, prostate, rectum, stomach, and cervical cancers each appeared in two pairs, and colon cancer in three.All of the 20 pairs shown in In this study we have comprehensively analyzed familial aggregation of cancer cases in a whole nation, both within and between pairs of cancer sites. The completeness of our genealogy database allows us to accurately trace distant relationships, which we believe is unique to this study. Linking the ICR to our nationwide genealogy database thus has made it possible to uncover distant familial connections between cancer cases, and reach beyond shared environmental factors to identify individual and combined cancer sites with the strongest genetic influences. Furthermore, even though the genetic effect decreases with more distant relationships, the sample sizes used to estimate familiality are dramatically larger for the distant relationships than for the closer ones. This compensates to some extent for the lower effect and adds considerable statistical power to the study.In this paper we restrict the presentation and discussion to the most significant findings. However, we provide results for all pairs of 27 cancer sites in The largest population-based studies reported to date, evaluating familial clustering within the same cancer site, are from Utah and Sweden ,9,10. ThAs seen in Utah and Sweden, high RR values were found in this study for multiple myeloma, lymphoid leukemia, and thyroid, testicular, and laryngeal cancer. The RR for thyroid cancer in first-degree relatives was much higher in Utah and Sweden (8.48 and 9.51) than in Iceland (3.02). One possible explanation of the lower RR may be the high incidence of thyroid cancer in Iceland, due to an excess of the papillary subtype ,37, whicThe cancer sites showing the highest RR for first-degree relatives tend to be among the rarer sites. There are two potential reasons why rare tumors tend to show higher RRs than common cancers. Being common, the baseline frequency is not low and that creates a bound on how large the RR can be. Also, common cancers are expected to be genetically complex, whereas it is more likely for a rare tumor to be closer to a Mendelian trait, caused by rare alleles with high penetrances.p values (after adjustment for multiple testing) for the combined group of first- to fifth-degree relatives. Furthermore, eight of these 16 sites remained significant even after exclusion of the first- and second-degree relatives (after adjustment for multiple testing). The majority of the 16 significant cancer sites are among the sites of the most prevalent cancers, indicating that we may lack power to detect extended familiality for the less prevalent cancer sites. Indeed the median number of cases per cancer site was 943 for the 16 significant sites compared to 342 for the non-significant sites. Nevertheless, significant familial clustering (first- to fifth-degree relatives) is seen for some of the less prevalent sites, i.e., lymphoid leukemia and esophagus and meningeal cancer.Most individual cancer sites, or 16 out of the 27 studied here, showed familiality as evidenced by significant The largest cancer twin study reported to date  documentA significantly increased risk of the same cancer was seen in mates only for individuals diagnosed with stomach, lung, or colon cancer. These results are in accordance with previous reports, including Swedish population-based studies, except for colon cancer ,39,40,41p values corresponding to the increased risk for first- to fifth-degree relationships. With this method, we detected 17 cancers that linked into 20 pairs of sites that were significant after adjustment for multiple testing. Stomach and prostate cancer appeared more frequently in the pairs than other cancer types, followed by colon, ovarian, and cervical cancer. We emphasize again, as with the same-cancer calculations, that we might lack power to connect rare cancers to other cancer sites. This possibility is highlighted by the fact that the 17 cancers in the significant pairs are the most prevalent cancer sites in Iceland.We also assessed the significance of familial clustering between cancer sites by calculating combined BRCA1 and BRCA2 may explain in part the cluster seen between prostate, breast, ovarian, and possibly pancreatic cancer [Some connections seen here between cancer sites may be partly explained by known high-risk genes involved in heritable syndromes. Thus, mutations in genes associated with hereditary nonpolyposis colorectal cancers could explain a part of the risk shared between stomach, colon, rectal, and endometrial cancer, and possibly brain and ovarian cancer ,43. In ac cancer ,44,45,46Undiscovered genetic factors could contribute to some connections seen here to a much greater extent than the known susceptibility factors. Although these could include unknown high-risk susceptibility genes, they are more likely multiple genetic variants, each conferring small to moderate risk.Familial clusters were identified between cancer sites, both in close and distant relatives, that do not correspond to known cancer syndromes. These include lung, esophageal, cervical, and stomach cancer, which, interestingly, have been associated with environmental rather than genetic factors. One explanation for this excess familiality between these cancer sites is an interaction of genetic susceptibility factors with environmental carcinogens (e.g., tobacco and diet) or infectious agents. Thus, the same environmental factor could interact with the same genetic susceptibility factor or factors to induce different cancers . Alternatively, different environmental factors could interact with the same genetic susceptibility factor or factors to increase the risk for different cancers .BRCA1 and BRCA2 mutations [Hormone-related cancers form another risk cluster. Thus, shared genetic susceptibility factors could directly influence the hormonal metabolism to induce breast, prostate, thyroid, or ovarian cancer in carriers. Alternatively, shared genetic factors could interact with dietary factors to induce aggregation of cancers at these sites in related individuals. A significantly increased risk of breast, prostate, cervical, and non-melanoma skin cancer was recently reported in first-degree relatives of early-onset breast cancer patients from Sweden that tested negative for utations . Our datTwo more groups of cancers with shared risk were identified that each include sites that share the same developmental progenitors: the prostate, kidney, and bladder are sites derived from the nephrogenic ridge while colon, rectum, and stomach are derived from the primitive gut tube. Therefore, the sites in each group may share risk alleles that regulate embryonic development, which can later play a role in oncogenesis.Interestingly, three cancer sites/types, non-melanoma skin, brain, and melanoma, that do not have significant same-cancer familial clustering demonstrate significant cross-cancer familial clustering with more prevalent cancer sites, i.e., rectum, stomach, and kidney cancers, respectively.Previous reports systematically evaluating the significance of co-clustering of cancer pairs in families have utilized the Utah Population Database. In these studies lip and prostate cancers appear to associate most frequently with other cancer sites. The same is true for prostate cancer in our study, whereas lip cancer does not significantly associate with any other cancer sites. This can at least in part be explained by the difference in age-standardized incidence rates for lip cancer in Iceland and Utah (Iceland 1.1 and Utah 2.4) . In contBecause the increased cross-site RR extends beyond the nuclear family, shared genetic factors may contribute to the risk of more than one cancer type. This suggests that cancer could be considered a broad phenotype with shared genetic factors across cancer sites. Therefore cancer should in certain cases be studied in a broader context than previously done. Combining multiple cancers that show increased cross-site RR may serve to increase the power of linkage and case-control studies. Our results also have implications for genetic counseling and imply that the focus of attention should broaden to the history of multiple cancer types in relatives within and outside the nuclear family. These results also suggest the utility of comparing expression profiles and in vitro biological processes across the cancers that we have identified as sharing genetic risk. The isolation of cancer predisposition genes with broad effects may define new rate-limiting pathways that can be used to search for drug targets for a more focused treatment with fewer side effects but with utility across multiple cancers.Table S1(1.9 MB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/projects/LocusLink/) accession numbers for the genes discussed in this paper are BRCA1(LocusLink ID 672) and BRCA2 (LocusLink ID 675).The LocusLink (Although a few cancers have a fairly simple genetic cause, most, especially the most common cancers, do not, and what makes one person rather than another develop cancer is not clear. One way of trying to work out how much genes rather than environment contribute to disease is to study large populations. One such population is the Icelandic nation: not only is detailed health information about individuals available, including information on cancer, but also very good genealogical information and a substantial amount of genetic data.Researchers examined all cancer records dating back to 1955 and then analyzed the chances of relatives and mates of these patients having cancer. They found that some cancers, especially rare ones, had a higher than baseline chance of occurring in relatives, but so did many common cancers, and for some cancers, the higher chances extended to quite distant relatives. In addition, the risk sometimes involved different cancer types.Even for the highest risk cancers, the absolute increased risk for relatives remains very small. In addition, despite the large numbers of patients studied, the numbers of cancer cases are still not large enough to be completely certain of the results, apart from very common cancers, which had the lowest chance of occurring in relatives. So these results will not help doctors much at the present time in telling an individual patient what their risk is of getting cancer if a relative has it—but they will be useful for other researchers in knowing how to plan future studies to look at the underlying causes of cancer.http://www.krabb.is/cancer/Icelandic Cancer Society: http://cis.nci.nih.gov/The United States National Cancer Institute's Cancer Information Service: http://www.cancerhelp.org.uk/CancerHelp UK, a free information service about cancer and cancer care: http://www.decode.comdeCODE Genetics:


Many primary malignancies spread via lymphatic dissemination, and accurate staging therefore still relies on surgical exploration. The purpose of this study was to explore the possibility of semiautomated noninvasive nodal cancer staging using a nanoparticle-enhanced lymphotropic magnetic resonance imaging (LMRI) technique.We measured magnetic tissue parameters of cancer metastases and normal unmatched lymph nodes by noninvasive LMRI using a learning dataset consisting of 97 histologically proven nodes. We then prospectively tested the accuracy of these parameters against 216 histologically validated lymph nodes from 34 patients with primary cancers, in semiautomated fashion. We found unique magnetic tissue parameters that accurately distinguished metastatic from normal nodes with an overall sensitivity of 98% and specificity of 92%. The parameters could be applied to datasets in a semiautomated fashion and be used for three-dimensional reconstruction of complete nodal anatomy for different primary cancers.These results suggest for the first time the feasibility of semiautomated nodal cancer staging by noninvasive imaging. Injection of targeted nanoparticles, combined with magnetic resonance imaging, allows identification and detailed three dimensional placement of malignant lymph nodes Most primary malignancies spread systemically via lymphatic dissemination . For exaNoninvasive imaging studies are commonly used during the workup of primary malignancies. Typically, lymph nodes are diagnosed by tomographic techniques (computed tomography [CT], magnetic resonance imaging [MRI]) as malignant when their short axis is >10 mm in size . Such siDespite the advances of LMRI for cancer staging, image analysis has been challenging and occasionally controversial. Traditional analysis has been based on a reader's identification of certain structural abnormalities that can be variable, given differences in acquisition parameters and interpretation criteria ,20,21. FThe goal of the current study was to develop and test technologies that would vastly improve the accuracy of current LMRI nodal staging. Specifically we set out to (a) determine whether unique magnetic parameters existed and could be used for semiautomated image analysis and (b) whether the technique could be applied to different primary cancers. Here we provide the first comprehensive analysis of tissue parameters validated against histopathology as an end point.n = 97 lymph nodes with known histopathology) and a test dataset  and/or nodal biopsy (n = 10). The investigated nodes had a mean short axis diameter of 10.5 mm (range 3–39 mm).The Institutional Review Board approved the current study and all patients signed informed consent. The study was divided into a learning (n = 18), breast (n = 7), penile (n = 4), bladder (n = 2), testes (n = 2), and colon (n = 1). Seventy-nine percent of the nodes were benign and 21% of the nodes were malignant. The nodes in the test dataset had a mean short axis diameter of 10.0 mm (range 3–39 mm). Both datasets included the full spectrum of normal nodes to completely replaced nodes.The test dataset was obtained from 34 patients  with histologically proven malignancies from different primaries , includin = 56) extended from the pubic symphysis to just above the level of aortic bifurcation. In patients with primary testicular cancers (n = 7) imaging was extended superiorly to include the renal hilum and retroperitoneum. In patients with breast cancer (n = 7) we obtained MR images of the bilateral axillae, including the internal mammary and supraclavicular regions. All patients were imaged with identical pulse sequences and timing parameters. Imaging was performed before and 24 h after intravenous ferumoxtran-10 administration .MRI was performed at 1.5 T  using phased-array coils. All images were archived on DICOM PACS servers  for subsequent analysis. Images of the pelvis (The acquired pulse sequences included (a) axial T2-weighted fast spin-echo ; (b) a T1-weighted two-dimensional gradient-echo sequence obtained in different anatomical planes ; (c) an axial T2-weighted dual TE gradient-echo ; and (d) a three-dimensional (3D) T1-weighted gradient echo sequence; TR/TE 4.5–5.5/1.4; flip angle, 15°; field of view, 24–28 cm; slice thickness, 1.4 mm; matrix, 256 × 256; in-plane resolution, 1.0 mm).The above listed imaging sequences and parameters had previously been optimized to reduce motion artifacts, maximize signal-to-noise ratio (SNR), and provide diagnostically useful images of the pelvis, abdomen, and chest within clinically acceptable time limits. The T2-weighted fast spin-echo sequence, in (a) above, was primarily used for qualitative nodal detection, and hence a square pixel with more than one acquisition was obtained. The two-dimensional axial T1-weighted gradient-echo sequence, in (b) above, was chosen to achieve adequate anatomical coverage within a short imaging time. The axial dual-echo gradient-echo sequence, in (c) above, was developed specifically for this project to provide artifact-free datasets for quantitative image analysis. A matrix size of 160 × 256 was chosen for this sequence to achieve a balance between the upper limits for imaging time while reducing image noise. Finally, a 3D T1-weighted sequence was obtained, in (d) above to provide a dataset for vascular maximum intensity projection (MIP) reconstructions.All image analysis was performed on archived DICOM images using different software packages . Lymph nodes were identified by readers who manually placed kernels onto each node for automated boundary detection and calculation of nodal dimensions and volumes. The thus identified regions of interest (ROIs) encompassed the entire lymph node (not only portions of it) and were used for quantitative signal-intensity (SI) measurements see . Serial noise. The T2* was calculated in nodal ROIs on dual TE images using CMIR-Image. T2* maps were constructed by performing fits of a standard exponential relaxation model (S = Ke–TE/T2*) to the data on a pixel-by-pixel basis. Only pixels with intensity greater than a threshold level (2X of noise) were considered during the fitting process. Pixel variance was obtained from post-MR images. Comparative visual analysis included short axis measurements, and identification of heterogeneity, large focal defects, and central hyperintensity, according to criteria previously established [A number of quantitative tissue parameters were calculated either as differences between pre- and postcontrast scans (δ) or as single-value analysis on postcontrast scans see . The lymablished ,17.To determine the diagnostic accuracy of the different tissue parameters in the learning dataset, we determined sensitivity, specificity, and predictive values for each parameter alone and in combination . The mosIn the final set of semiautomated image analysis, 3D reconstructions were obtained for nodal mapping onto vascular anatomy using MIP projections. While the MIP projections do not aid in the differentiation between malignant and benign lymph nodes, they are invaluable in providing anatomic content to the dozens of lymph nodes identified. In particular, MIP images were generated interactively from postcontrast, fat-saturated, volumetric interpolated breath-hold images to outline vascular anatomy. The evaluated lymph nodes characterized as benign or malignant  were then superimposed on the volumetric 3D images, using customized software .t-test , and values are summarized in Data were expressed as mean ± standard deviations (SD) and medians. All statistical testing was performed using GraphPad Prism . The significance between two individual groups was determined using the nonpaired Student's n = 55; both benign and malignant nodes) or in fine needle aspirates and core biopsies , implementing careful mapping procedures to correlate nodes. Surgically excised nodes were sectioned at 10–20 μm intervals after bihalving and were stained with hematoxylin-eosin.All lymph nodes were sampled histologically within 2 wk of the MRI . The analysis was done in surgically resected lymph nodes  and specificity  in the learning dataset E. Using To determine whether feature extraction would be accurate for prospective nodal staging, we utilized the above criteria against a larger test dataset encompassing 216 validated lymph nodes from 34 patients, including different primaries see . The senUsing the dual-value analysis, there were two malignant outliers in the benign dataset  and three benign outliers in the malignant dataset . More important, all the misclassified nodes occurred in individual patients rather than in the same patient and, hence, did not affect the overall nodal staging on a patient-by-patient basis in this dataset.Utilizing semiautomated feature extraction to identify lymph nodes and image analysis (based on T2* and pixel variance), we subsequently proceeded to map individual lymph nodes onto vascular anatomy in the different anatomic drainage patterns. We show that it is feasible to extract various quantitative tissue parameters to predict the likelihood of nodal metastases in vivo. These results are highly relevant in cancer staging because they provide evidence that (a) quantitative tissue parameters enable diagnosis of lymph node metastases while reducing interobserver variability and (b) that semiautomated reconstructions allow spatially more extensive mapping than is currently possible.Metastases to lymph nodes occur during growth of most primary malignancies, and their presence mandates the need for more extensive and systemic therapy. Nodal cancer staging currently relies on invasive procedures  with significant morbidity and cost ,23, or iThe significance of the above findings is 3-fold. First, the ability to directly and noninvasively monitor nodal tumor involvement represents a powerful diagnostic tool for cancer. Accurate staging represents the cornerstone for triaging patients to either localized or to more aggressive and systemic therapies. Second, the method described here was sensitive for the limited subsets of primary cancers tested. It is reasonable to hypothesize that such analysis could be applied to staging of other common primaries. In particular, lung, colorectal, genitourinary, and head and neck cancers could benefit from this staging procedure. In addition to nodal staging, the nanoparticle-enhanced MRI can also be used to measure microvascularity in primary tumors  and to iThe LMRI staging technique is believed to be clinically relevant in several key areas. First, LMRI may play a significant role in avoiding unnecessary surgeries, that is, those in node-positive patients. Second, since LMRI can detect lymph nodes outside traditional surgical fields, this information may influence surgical approaches. In colorectal cancer, LMRI may provide a “sentinel-node-like” guide to staging. Third, it is likely that LMRI would be useful to identify appropriate patients to receive neoadjuvant chemotherapy prior to surgery. Currently, neoadjuvant therapy is often reserved for postoperative patients, once the nodal status has been determined. Fourth, LMRI may be particularly useful to guide radiation therapy by mapping the complete nodal status onto bony and vascular landmarks. Finally, LMRI could be used to avoid invasive diagnostic procedures, which are not part of therapy. For example, LMRI could replace lymphangiography, mediastinoscopy, or endoscopic ultrasound for nodal staging.Our findings have a number of direct implications for technology development and in clinical care. Accurate measurements of T2* relies on motion artifact-free multiecho pulse sequences that are not routinely available on clinical scanners at spatial resolutions required for nodal staging. Such sequences will have to be implemented and combined with postprocessing tools to simplify and semiautomate analysis. Similar software approaches are already used routinely in lung nodule characterization  or screeWhen deciding on treatment for patients with cancer, it is very important to assess whether the cancer has spread to lymph nodes—both to help decide what treatment a patient should have and what the eventual outcome might be. Previous ways of finding involved lymph nodes included taking out the nodes by surgery, ultrasound, and CT and MRI scans.A solution of magnetic nanoparticles that tend to go to lymphoid organs was injected and then tracked by MRI. The pattern of the particles was abnormal when there was metastasis in the nodes, and it was possible to train a computer to recognize this abnormality. The authors developed the program in one group of patients and then tested it in another group, in which they were able to correctly predict whether the nodes were involved in about nine of ten nodes. In addition, they could use the information to display a virtual picture of the involved nodes.The technique will need to be validated in a larger group of patients, and by other investigators. However, it means that it is potentially possible to work out much more precisely, and with less chance of error, whether lymph nodes are involved in cancer. Hence, treatment can be better planned, and if surgery is needed to remove nodes for analysis, then this technique could ensure that the surgery is as minimal as possible.http://www.radiologyinfo.org/RadiologyInfo, a public information site developed by the American College of Radiology and the Radiological Society of North America: http://www.nlm.nih.gov/medlineplus/cancer.htmlMedline Plus, which has health information from the National Library of Medicine: