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Background: Bacteriophages (phages) have been proposed as an alternative to antibiotics and surface disinfectants for treatment of Flavobacterium psychrophilum biofilms and fish infections in aquaculture settings. The aim of the study was to estimate the minimal phage:host ratio (PHR) required for the control of in vitro biofilm formation and mortalities caused by F. psychrophilum in experimentally infected fish. Materials and Methods: F. psychrophilum cells in different stages of biofilm formation were exposed to the lytic phage FPSV-D22 at different PHRs. Results: Our results show that an initial PHR of 0.01 is sufficient for more than an 80% inhibition of attachment and colonization, and disruption of maturated F. psychrophilum biofilms, whereas greater ratios resulted in almost complete interruption of the different biofilm stages. Interestingly, a similar response was observed in a phage therapy trial with live rainbow trout (Oncorhynchus mykiss), where treatment of F. psychrophilum-infected fish by injection of serial bacteriophage doses resulted in significantly (***p ≤ 0.001) higher survival already at a PHR of 0.02. Conclusions: These results indicate that phages have the potential to be effective for control and treatment of F. psychrophilum infections in fish farms even when applied in concentrations lower than previously expected.

BackgroundRadiation-associated angiosarcoma of the breast (RAASB) is an aggressive malignancy that is increasing in incidence. Only a few previous population-based studies have reported the results of RAASB treatment.MethodsA search for RAASB patients was carried out in the Finnish Cancer Registry, and treatment data were collected to identify prognostic factors for survival.ResultsOverall, 50 RAASB patients were identified. The median follow-up time was 5.4 years (range 0.4–15.6), and the 5-year overall survival rate was 69%. Forty-seven (94%) patients were operated on with curative intent. Among these patients, the 5-year local recurrence-free survival, distant recurrence-free survival, and overall survival rates were 62%, 75%, and 74%, respectively. A larger planned surgical margin was associated with improved survival.ConclusionsWe found that the majority of RAASB patients were eligible for radical surgical management in this population-based analysis. With radical surgery, the prognosis is relatively good.

Increasing problems with antibiotic resistance have directed interest toward phage therapy in the aquaculture industry. However, phage resistance evolving in target bacteria is considered a challenge. To investigate how phage resistance influences the fish pathogen Flavobacterium columnare, two wild-type bacterial isolates, FCO-F2 and FCO-F9, were exposed to phages (FCO-F2 to FCOV-F2, FCOV-F5, and FCOV-F25, and FCO-F9 to FCL-2, FCOV-F13, and FCOV-F45), and resulting phenotypic and genetic changes in bacteria were analyzed. Bacterial viability first decreased in the exposure cultures but started to increase after 1 to 2 days, along with a change in colony morphology from original rhizoid to rough, leading to 98% prevalence of the rough morphotype. Twenty-four isolates (including four isolates from no-phage treatments) were further characterized for phage resistance, antibiotic susceptibility, motility, adhesion, and biofilm formation, protease activity, whole-genome sequencing, and virulence in rainbow trout fry. The rough isolates arising in phage exposure were phage resistant with low virulence, whereas rhizoid isolates maintained phage susceptibility and high virulence. Gliding motility and protease activity were also related to the phage susceptibility. Observed mutations in phage-resistant isolates were mostly located in genes encoding the type IX secretion system, a component of the Bacteroidetes gliding motility machinery. However, not all phage-resistant isolates had mutations, indicating that phage resistance in F. columnare is a multifactorial process, including both genetic mutations and changes in gene expression. Phage resistance may not, however, be a challenge for development of phage therapy against F. columnare infections since phage resistance is associated with decreases in bacterial virulence. IMPORTANCE Phage resistance of infectious bacteria is a common phenomenon posing challenges for the development of phage therapy. Along with a growing world population and the need for increased food production, constantly intensifying animal farming has to face increasing problems of infectious diseases. Columnaris disease, caused by Flavobacterium columnare, is a worldwide threat for salmonid fry and juvenile farming. Without antibiotic treatments, infections can lead to 100% mortality in a fish stock. Phage therapy of columnaris disease would reduce the development of antibiotic-resistant bacteria and antibiotic loads by the aquaculture industry, but phage-resistant bacterial isolates may become a risk. However, phenotypic and genetic characterization of phage-resistant F. columnare isolates in this study revealed that they are less virulent than phage-susceptible isolates and thus not a challenge for phage therapy against columnaris disease. This is valuable information for the fish farming industry globally when considering phage-based prevention and curing methods for F. columnare infections.

Phage-based approaches have gained increasing interest as sustainable alternative strategies to antibiotic treatment or as prophylactic measures against disease outbreaks in aquaculture. The potential of three methods (oral, bath, and injection) for delivering a two-component phage mixture to rainbow trout fry for controlling Flavobacterium psychrophilum infections and reduce fish mortality was investigated using bacteriophages FpV4 and FPSV-D22. For the oral administration experiment, bacteriophages were applied on feed pellets by spraying (1.6 × 108 PFU g–1) or by irreversible immobilization (8.3 × 107 PFU g–1), using the corona discharge technology (Fixed Phage Ltd.). The fish showed normal growth for every group and no mortality was observed prior to infection as well as in control groups during the infection. Constant detection of phages in the intestine (∼103 PFU mg–1) and more sporadic occurrence in kidney, spleen, and brain was observed. When fish were exposed to F. psychrophilum, no significant effect on fish survival, nor a direct impact on the number of phages in the sampled organs, were detected. Similarly, no significant increase in fish survival was detected when phages were delivered by bath (1st and 2nd bath: ∼106 PFU ml–1; 3rd bath: ∼105 PFU ml–1). However, when phages FpV4 and FPSV-D22 (1.7 × 108 PFU fish–1) were administered by intraperitoneal injection 3 days after the bacterial challenge, the final percent survival observed in the group injected with bacteriophages FpV4 and FPSV-D22 (80.0%) was significantly higher than in the control group (56.7%). The work demonstrates the delivery of phages to fish organs by oral administration, but also suggests that higher phage dosages than the tested ones may be needed on feed pellets to offer fish an adequate protection against F. psychrophilum infections.

Increasing problems with antibiotic resistance has directed interest towards phages as tools to treat bacterial infections in the aquaculture industry. However, phage resistance evolves rapidly in bacteria posing a challenge for successful phage therapy. To investigate phage resistance in the fish pathogenic bacterium Flavobacterium columnare, two phage-sensitive, virulent wild-type isolates, FCO-F2 and FCO-F9, were exposed to phages and subsequently analyzed for bacterial viability and colony morphology. Twenty-four phage-exposed isolates were further characterized for phage resistance, antibiotic susceptibility, motility, adhesion and biofilm formation on polystyrene surface, protease activity, whole genome sequencing and virulence against rainbow trout fry. Bacterial viability first decreased in the exposure cultures, subsequently increasing after 1-2 days. Simultaneously, the colony morphology of the phage-exposed isolates changed from original rhizoid to rough. The rough isolates arising in phage exposure were phage-resistant with low virulence, whereas rhizoid isolates maintained phage sensitivity, though reduced, and high virulence. Gliding motility and protease activity were also related to the phage sensitivity. Observed genetic mutations in phage-resistant isolates were mostly located in genes coding for type IX secretion system, a component of the flavobacterial gliding motility machinery. However, there were mutational differences between individual isolates, and not all phage-resistant isolates had genetic mutations. This indicates that development of phage resistance in F. columnare probably is a multifactorial process including both genetic mutations and changes in gene expression. Phage resistance may not, however, be a challenge for development of phage therapy against F. columnare infections, since phage resistance is associated with decrease in bacterial virulence.ImportancePhage resistance of infectious bacteria is a common phenomenon posing challenges for development of phage therapy. Along with growing World population and need for increased food production, constantly intensifying animal farming has to face increasing problems of infectious diseases. Columnaris disease, caused by F. columnare, is a worldwide threat for salmonid fry and juvenile farming. Without antibiotic treatments, infections can lead to 100% mortality in a fish stock. Phage therapy of columnaris disease would reduce a development of antibiotic-resistant bacteria and antibiotic loads by the aquaculture industry, but phage-resistant bacterial isolates may become a risk. However, phenotypic and genetic characterization of phage-resistant F. columnare isolates in this study revealed that they are less virulent than phage-sensitive isolates and thus not a challenge for phage therapy against columnaris disease. This is a valuable information for the fish farming industry globally when considering phage-based prevention and curing methods for F. columnare infections.

To investigate whether European whitefish (Coregonus lavaretus) groups in the brackish (≤7%) Gulf of Bothnia (Baltic Sea) express specific otolith elemental characteristics, concentrations of elements (Ca, Ba, Sr, Zn, Mn, Fe) in whole otoliths were studied by inductively coupled plasma-optical emission spectrometry. Whitefish (N = 67) were sampled from six different latitudinal sites; four in the sea and two in rivers along the west coast of Finland, and from a fresh water lake for comparison. The concentration of several elements showed significant differences among sampling sites. Otolith Ba concentrations were higher at the northern sampling sites compared to the southern ones, thereby showing negative association to capture-habitat salinity. In contrast, otolith Sr and Zn concentrations were lower at the northern sampling sites, thereby associating positively to capture-habitat salinity. Otolith Mn concentrations did not associate with latitude or salinity but markedly varied between sampling sites. Elevated Fe concentrations occurred in fish otoliths from the two northernmost sites. Ca concentrations were at the same levels in otoliths from all whitefish samples. Otoliths of whitefish from the lake had very high Ba and low Sr concentrations compared to concentrations in otoliths from whitefish in the sea and rivers. Our results show that otolith elemental characteristics of whitefish vary considerably at various geographic scales showing high potential for population identification using elemental fingerprints. Analysis of otolith elemental characteristics may therefore provide an efficient tool for the identification of main areas of residence, spawning locations, and migration routes, thus contributing towards the sustainable management of whitefish stocks.

The antimicrobial susceptibilities of TurkishF. psychrophilumisolates were investigated using a Clinical and Laboratory Standards Institute (CLSI) standard microdilution testing protocol. The significance of the minimum inhibitory concentrations was determined by applying empirically and statistically determined epidemiological cut-off values calculated by normalized resistance analysis. Twenty-fiveF. psychrophilumisolates made over four years from three different trout species in five different regions were examined. All isolates were fully susceptible to amoxicillin and florfenicol, but 88% of our isolates were categorized as having reduced susceptibility to the two quinolones, oxolinic acid, and enrofloxacin. Eighty percent of the isolates were categorized as showing reduced susceptibility to oxytetracycline.Sequence analysis demonstrated that the reduced susceptibility to the quinolones was associated with chromosomal mutations in thegyrA andparE genes. PCR analysis demonstrated that the genestetA,tetB,tetC,tetE,tetH,tetL, andtetM were not associated with the reduced susceptibility to oxytetracycline and the significance of this observation is discussed. PCR analysis also showed the presence ofsul2 in six isolates andfloR in one isolate. This study is the first report of these genes inF. psychrophilum.It was noted, however, that thefloR was detected in an isolate categorized as fully susceptible to florfenicol, but the reasons for its lack of expression in the phenotype was not investigated.It is argued that the Turkish trout industry, in common with those of many countries world-wide, is critically dependent on the continued susceptibility ofF. psychrophilumto florfenicol.

Flavobacterium psychrophilum isolated from rainbow trout Oncorhynchus mykiss suffering from bacterial cold-water disease (BCWD) can dissociate into 2 morphological colony types, rough (R) and smooth (S). However, the presence of the 2 morphotypes in disease outbreaks has not yet been investigated thoroughly. We examined the occurrence of R and S morphotypes in rainbow trout from BCWD outbreaks and in unfertilized eggs from a hatchery. The isolated colony types were characterized by pulsed field gel electrophoresis (PFGE), plasmid analysis, and oxolinic acid susceptibility testing. From most outbreaks, both morphotypes were isolated, although the S type only was isolated from the majority of individual fish. PFGE analysis showed both diverse and indistinguishable genetic patterns among the concurrent morphotypes. While PFGE patterns common to both fish and egg isolates were identified, this was not always the case. Resistant and sensitive isolates of both colony types were isolated from individual disease out- breaks. The plasmid pattern was partly associated with the colony type, showing identical or com- pletely different patterns for the R and S types isolated from the same outbreak. The study showed that within a BCWD outbreak, F. psychrophilum cells with different morphology, plasmid content, antibiotic susceptibility, and PFGE pattern can be isolated, suggesting that F. psychrophilum populations infecting rainbow trout in farm environments can be diverse and thus complicate the control of the disease.

The adherence of Flavobacterium psychrophilum to surfaces of epithelial tissues has been inconclusively suggested as a mechanism, which enables the bacterium to invade the host. Hence, the present study aimed to examine the adherence of the cells of two colony phenotypes, smooth and rough, of F. psychrophilum to mucosal tissues of rainbow trout fry and to test the skin mucus as a nutrient for the growth of F. psychrophilum. Fish were immersed in water containing 106 CFU mL-1 F. psychrophilum for each colony phenotype. Mucosal tissue samples from fins, gills, skin and eyes, and swab samples from spleen and kidney were taken and inoculated onto TYES agar plates. Colony phenotypes of F. psychrophilum were identified and number of colonies counted. The results showed that cells of both phenotypes initially (0 h) adhered to all mucosal surfaces, but only the rough cells were still present on tissues 1 h post-immersion. Both phenotypes showed a tissue tropism with the fin tissue being the most adhered. Furthermore, skin mucus promoted the growth of both colony phenotypes. We suggest that the growth of F. psychrophilum cells in skin mucus apparently facilitates the bacterial adherence to mucosal surfaces, and the subsequent invasion into the host.

Treatment of bacterial fish diseases can be complicated by resistant bacterial biofilms harbouring pathogenic bacteria and causing recurrent exposure of fish to infections. In this study, the effect of biofilm formation on antimicrobial tolerance was examined using three bacterial isolates of the fish pathogen Flavobacterium psychrophilum and two antimicrobial agents, oxytetracycline and flumequine, commonly used in aquaculture. Planktonic and biofilm cells were exposed to a minimum inhibitory concentration (MIC), to a 3 × MIC concentration and to an environmental concentration level of each antimicrobial in 96-well microtitre plates after which growth on agar plates was measured. The type strain NCIMB1947 of F. psychrophilum was further used to study the development of antimicrobial resistance in biofilm cells. The results suggest that at high bacterial densities (>10(7) CFU mL(-1)), biofilm cells of F. psychrophilum are less susceptible to antimicrobial agents. Furthermore, the results imply that biofilm cells of F. psychrophilum may rapidly develop resistance to both oxytetracycline and flumequine if exposed to subinhibitory concentrations of these antimicrobials.

From March 1994 to March 1998, breast cancer patients (an estimated relapse risk with 70% or more within five years with standard therapy) were randomised to treatment with tailored fluorouracil, epirubicin, and cyclophosphamide (FEC) therapy or FEC followed by marrow-supported high dose therapy in the Scandinavian Breast Group 9401 study. The aim of the present paper was to investigate differences in toxicity and eight health-related quality of life (HRQoL) variables (physical functioning, role functioning, emotional functioning, social functioning, cognitive functioning, fatigue, nausea-vomiting, and global quality of life) between women in the six dose steps used in the tailored and granulocyte colony stimulating factor supported FEC-arm at the assessment point 16 weeks after random assignment to treatment.The European Organization and Treatment of Cancer Quality of Life Questionnaire EORTC QLQ-C30 were mailed to the patients.A total of 157 (87%) in the tailored FEC-group responded to the questionnaire within the time frame 16 weeks after inclusion in the study. Overall, toxicity was low, reaching grade 1-2 also in the higher dose steps. There were no overall differences between the dose steps on any of the tested HRQoL variables. Patients at dose step 4 scored statistically significantly higher on physical functioning than patients at dose step 1 (p = 0.022) and compared to those at dose step 2 (p = 0.014). Patients at dose steps -2 and -1 (combined to one group) reported statistically significantly higher mean scores on cognitive functioning than patients at dose step 1 (p = 0.022).Patients who received higher doses, based on the tailored dosing strategy, did not seem to have worse HRQoL than those who had lower doses.

Based on randomized studies bone-marrow supported (BMS) high-dose chemotherapy (HDCT) is not superior to conventional CT as adjuvant treatment for high-risk breast cancer. To compare the cost-effectiveness of these treatments we examined the data of Finnish patients in the SBG9401 trial 1. Patients were randomized to receive either dose-escalated (de FEC) (group A, n =59) or FEC and HDCT+BMS (group B, n =70). They received adjuvant radiotherapy (RT) + tamoxifen. All direct health care costs of first line treatment at the oncology units were considered as well as productivity costs within the first 3 years of follow-up. Effectiveness was measured by the number of survival days during 5 years of follow-up. The mean direct health care costs were significantly higher in group B (25829 euro in group A vs. 36605 euro in group B, p0.001), mainly due to a higher number of hospital days. Half of the costs in group A was due to the use of filgrastim (15335 euro in A and 2969 euro in B, p0.001). The costs of RT were only 5% of total costs. There was no statistically significant difference between the groups in the number of survival days, but sensitivity analysis based on bootstrapping suggested that treatment A would be a less costly and more effective alternative in a great majority of cases.

Purpose Amplification of the HER-2/neu and topoisomerase IIα (TOP2A) genes has been linked to the effects of anthracyclines. Their role in predicting the outcome of anthracycline-based adjuvant chemotherapy for breast cancer patients has remained controversial. Patients and Methods The present substudy of the Scandinavian Breast Group trial 9401, in which an epirubicin-based regimen (nine courses of tailored and dose-escalated fluorouracil, epirubicin, and cyclophosphamide [FEC]) was compared with three or four courses of standard FEC followed by bone marrow–supported high-dose chemotherapy (cyclophosphamide, thiotepa, and carboplatin), included high-risk breast cancer patients (with eight or more positive axillary lymph nodes or at least five nodes with additional poor prognostic indicators). Amplification of HER-2/neu was determined retrospectively in paraffin-embedded tumor tissue sections by chromogenic in situ hybridization. TOP2A was tested only in HER-2/neu–amplified tumors. Results HER-2/neu amplification alone, which was present in 32.7% of the tumors, was a strong prognostic factor for short relapse-free (P = .0034) and overall survival (P = .0008) but showed no direct association with treatment assignment. TOP2A coamplification, which was present in 37% of HER-2/neu–amplified tumors, was associated with better relapse-free survival in patients treated with tailored and dose-escalated FEC (hazard ratio [HR] = 0.45; P = .049). A statistical multivariate Cox's regression analysis confirmed the predictive significance of TOP2A coamplification (HR = 0.30; P = .020) in HER-2/neu–amplified tumors. There was no such association in patients with HER-2/neu–amplified tumors without TOP2A gene amplification. Conclusion Coamplification of HER-2/neu and TOP2A may define a subgroup of high-risk breast cancer patients who benefit from individually tailored and dose-escalated adjuvant anthracyclines.

Invasive fungal infections (IFI) constitute a majorcomplication in allogeneic stem cell transplant(SCT) recipients, with an incidence of about 15%(1, 2). During the recent years, Aspergillus infec-tions have been predominant in this setting (1–4).In autologous stem cell transplant (ASCT)recipients, the risk of IFI has been considered tobe small when compared with allogeneic patients(5, 6). Several small single-centre series have shownan incidence of 1–2% or even less (7–11), althoughhigher figures have been observed especially inpatients receiving bone marrow support instead ofblood stem cells (12, 13). Recently, an incidence upto 5% for invasive aspergillosis (IA) in an autol-ogous setting has been reported from Seattle (14).The risk of Candida infections in autologous bonemarrow transplant recipients has been high in someearlier studies (15, 16).Several changes in autologous transplant pro-cedure have occurred during the last decade.These include the use of blood stem cells insteadof marrow cells, the use of granulocyte-colony-stimulating factor (G-CSF) after stem cell infu-sion, the use of manipulated stem cell grafts andfluconazole prophylaxis. In addition, improveddiagnosis of Aspergillus infections, includingJantunen E, Salonen J, Juvonen E, Koivunen E, Siitonen T, Lehtinen T,Kuittinen O, Leppa¨ S, Anttila V-J, Ita¨la¨ M, Wiklund T, Remes K,Nousiainen T. Invasive fungal infections in autologous stem celltransplant recipients: a nation-wide study of 1188 transplanted patients.Eur J Haematol 2004: 73: 174–178. Blackwell Munksgaard 2004.Abstract: Based on small single-centre series, the risk of invasive fungalinfections (IFI) has been considered small in autologous stem celltransplant (ASCT) recipients. Purpose: To analyse epidemiologicaland clinical features of (IFI) among ASCT recipients in Finland 1990–2001. Patients: During the study period, 1188 adult patients receivedhigh-dose therapy supported by ASCT in six centres. Altogether, 1112patients (94%) received blood progenitor cells. The graft was CD34+selected in 261 patients (22%). The major diagnostic groups werenon-Hodgkin’s lymphoma (n ¼ 417), multiple myeloma (n ¼ 395),breast cancer (n ¼ 132) and Hodgkin’s lymphoma (n ¼ 53).Results: Eighteen patients (1.5%) with IFI were identified. The inci-dence of proven or probable invasive aspergillosis was 0.8%, followedby candidaemia with an incidence of 0.3%. The median time to thediagnosis of IFI was 35 d (6–162) from the progenitor cell infusion. Infourteen patients (78%) IFI was diagnosed during lifetime and they weretreated with antifungal therapy for a median of 50 d. Nine patients(64%) were cured. Conclusions: IFI appears to be a rare event afterASCT and Aspergillus infections seem to be predominant. These epi-demiological features have an impact in planning prophylactic andempirical antifungal strategies in ASCT recipients.E. Jantunen

Limited experience is available on the feasibility and efficacy of high-dose therapy (HDT) supported by autologous stem cell transplantation (ASCT) in patients with peripheral T-cell lymphoma (PTCL). Therefore, a nation-wide survey was conducted in adult patients transplanted for PTCL in Finland during 1990-2001. After histopathology review, 37 patients were identified. The median age was 46 years (16-68) at the time of ASCT. Histology included PTCL not otherwise specified in 14 patients, anaplastic large cell lymphoma (ALCL) in 14 patients, and other in nine patients. Disease status at the time of ASCT was CR/PR1 in 18 patients; CR/PR2 in 14 patients, and other in five patients. HDT consisted of either BEAC (N=22) or BEAM (N=15), supported by blood stem cells in 34 patients (92%). Early transplant-related mortality was 11%. With a median follow-up of 24 months from HDT, 16 patients (43%) have relapsed or progressed. The estimated 5-year overall survival (OS) was 54%. Patients with ALCL had superior OS when compared with other subtypes (85 vs 35%, P=0.007). OS at 5 years was 63% in patients transplanted in CR/PR1 vs 45% in those transplanted in other disease status (P=NS). Prospective studies are needed to define the role of ASCT in this lymphoma type.

From 1990 to 1997, 113 eligible patients with classical osteosarcoma received neo-adjuvant chemotherapy consisting of high-dose methotrexate, cisplatin and doxorubicin. Good histological responders continued to receive the same therapy postoperatively, while poor responders received salvage therapy with an etoposide/ifosfamide combination. With a median follow-up of 83 months, the projected metastasis-free and overall survival rates at 5 years are 63 and 74%, respectively. Independent favourable prognostic factors for outcome were tumour volume 4.5 μM and female gender. The etoposide/ifosfamide replacement combination did not improve outcome in the poor histological responders. In conclusion, this intensive multi-agent chemotherapy results in >70% of patients with classical osteosarcoma surviving for 5 years. The data obtained from this non-randomised study do not support discontinuation and exchange of all drugs used preoperatively in histological poor responders. As observed in previous Scandinavian osteosarcoma studies, female gender appears to be a strong predictor of a favourable outcome.

ContextSecond cancer is the leading cause of death in long-term survivors of Hodgkin disease (HD), with exceptionally high risks of breast cancer among women treated at a young age. Quantitative associations between radiotherapy dose delivered to the breast and administered chemotherapy have not been reported to date in large series, nor has the influence of ovarian exposures on subsequent risk.ObjectiveTo quantify the long-term risk of breast cancer associated with use of radiotherapy and chemotherapy to treat young women with HD.Design, Setting, and SubjectsMatched case-control study of breast cancer within a cohort of 3817 female 1-year survivors of HD diagnosed at age 30 years or younger, between January 1, 1965, and December 31, 1994, and within 6 population-based cancer registries. The study was conducted March 1, 1996, through September 30, 1998.Main Outcome MeasuresRelative risk (RR) of breast cancer associated with radiation dose delivered to site of breast cancer or to ovaries and with cumulative dose of alkylating agents.ResultsBreast cancer occurred in 105 patients with HD who were matched to 266 patients with HD but without breast cancer. A radiation dose of 4 Gy or more delivered to the breast was associated with a 3.2-fold (95% confidence interval [CI], 1.4-8.2) increased risk, compared with the risk in patients who received lower doses and no alkylating agents. Risk increased to 8-fold (95% CI, 2.6-26.4) with a dose of more than 40 Gy (P

Purpose: Pulmonary toxicity was prospectively evaluated within a randomized trial for breast cancer patients at high risk for relapse, who postoperatively received as adjuvant therapy either 9 cycles of tailored chemotherapy (20 patients) (cyclophosphamide, epirubicin, 5-fluorouracil [FEC]) or standard FEC × 3 followed by high-dose chemotherapy (cyclophosphamide, thiotepa, carboplatin [CTCb]) supported by peripheral blood stem cell transplantation (14 patients). After high-dose chemotherapy or tailored FEC, all patients received locoregional radiotherapy (50 Gy/5 weeks), plus tamoxifen for 5 years. Methods and Materials: Lung function tests (FVC, FEV1, and DL CO ) were performed before chemotherapy and 9 months after radiotherapy. Computed tomography of the lungs was performed before radiotherapy and 6 weeks, 3 months, and 9 months after radiotherapy. Results: Clinical signs of suspected pneumonitis were noted in 29% of patients, but only 1 patient needed symptomatic therapy. Radiologic changes were detected in 68% of patients, and they were most frequent at 3 months after radiotherapy. FVC decreased in both groups (tailored FEC: mean difference, −6.5%, p = 0.0005; CTCb: −2.0%, p = 0.21; tailored FEC vs. CTCb: −4.5%, p = 0.05). DL CO decreased significantly in both groups (tailored FEC: mean difference, −11.2%, p p = 0.02; tailored FEC vs. CTCb: −5.6%, p = 0.07). FEV1 decreased by 7.3% in patients treated with tailored FEC ( p p = 0.03) (tailored FEC vs. CTCb: 3.7%, p = 0.08). Conclusions: Changes in pulmonary function were thus detected in both groups, although to a greater extent in the tailored FEC group. The clinical significance of these findings should be balanced carefully against the improved, statistically significant relapse-free survival achieved with the tailored FEC regimen compared to high-dose CTCb + peripheral blood stem cell transplantation (PSCT).

This report is based on 1.851 adult patients with soft tissue sarcoma (STS) of the extremities or trunk wall diagnosed between 1986 and 1997 and reported from all tertiary referral centers in Norway and Sweden. The median age at diagnosis was 65 years and the male-to-female ratio was 1.1:1. One third of the tumors were subcutaneous, one third deep, intramuscular and one third deep, extramuscular. The median size was 7 (1-35) cm and 75% were high grade (III-IV). Metastases at presentation were diagnosed in 8% of the patients. Two thirds of STS patients were referred before surgery and the referral practices have improved during the study. The preoperative morphologic diagnosis was made with fine-needle aspiration cytology in 81%, core-needle biopsy in 9% and incisional biopsy in 10%. The frequency of amputations has decreased from 15% in 198688 to 9% in 1995-1997. A wide surgical margin was achieved in 77% of subcutaneous and 60% of deep-seated lesions. Overall, 24% of operated STS patients had adjuvant radiotherapy. The use of such therapy at sarcoma centers increased from 20% 1986-88 to 30% in 1995-97. Follow-up has been reported in 96% of the patients. The cumulative local recurrence rate was 0.20 at 5 years and 0.24 at 10 years. The 5-year metastasis-free survival rate was 0.70.

BACKGROUND. Radiotherapy is a known risk factor for sarcoma development. Postirradiation sarcomas arise within the radiation field after a latency period of several years and usually are highly malignant. Very little is yet known about their genetic changes. METHODS. Twenty-seven postirradiation sarcomas were analyzed by comparative genomic hybridization, which allows genome-wide screening of DNA sequence copy number changes. RESULTS. Copy-number aberrations were detected in 20 (74%) tumors. The mean number of aberrations per tumor was 5.3 with gains outnumbering losses. The most frequent gains affected the minimal common regions of 7q11.2-q21 and 7q22 in 30% and 7p15-pter in 26%. Gain of 8q23-qter was detected in 22%. The most frequent losses affected 11q23-qter and 13q22-q32 in 22%. In osteosarcomas, the most frequent aberration was loss of 1p21-p31, in malignant fibrous histiocytomas (MFH) gain of 7cen-q22, and in fibrosarcomas gain of 7q22. The findings in postirradiation osteosarcomas and MFHs were compared with findings in sporadic osteosarcomas and MFHs, reported previously by the authors. In sporadic osteosarcomas, gains outnumbered losses, but, in postirradiation osteosarcomas, losses were more frequent than gains. Loss at 1p was rare in sporadic osteosarcoma (3%) but frequent (57%) in postirradiation osteosarcomas. Gains at 7q were frequent both in postirradiation and sporadic MFH. CONCLUSIONS. According to previous studies on different types of sporadic sarcomas, gains at 7q or 8q are associated with poor prognosis or large tumor size. Thus, the frequent gains at 7q and 8q might have been responsible in part for the poor prognosis of postirradiation sarcomas. Also, however, some of their clinical features, i.e., high malignancy grade, late diagnosis, and central location, are associated with a poor prognosis.

In many malignant diseases the expression levels of CD44 and its splice variant v6 (CD44v6) have been associated with the prognosis. The purpose of this study was to investigate the clinical significance of CD44 in adult soft tissue sarcomas (STS). 133 STS patients with a limb or superficial trunk tumour treated at the Helsinki University Central Hospital in 1987–1993 with a median follow-up time of 68 months were included in this study. The expression of CD44 and CD44v6 was determined immunohistochemically on paraffin-embedded tumour samples. 95% of the tumours expressed CD44 and CD44v6 was detected in 57%. Strong CD44 expression was associated with low grade (P = 0.04) and small tumour size (P = 0.02). In diploid tumours the CD44 expression was correlated with low S-phase fraction (P = 0.001). High expression of both, CD44 in general as well as that of CD44v6, predicted a higher risk for local recurrence (CD44: P = 0.01 and CD44v6: P = 0.05). Low CD44v6 content of the primary tumour correlated with poor survival (P = 0.02). Determining the expression of CD44 or CD44v6 in a primary STS could be a valuable tool for selecting the group of patients who might benefit from intensified local tumour treatment. © 2001 Cancer Research Campaign http://www.bjcancer.com

Rituximab (IDEC-C2B8, Mabthera, Rituxan), a chimeric monoclonal antibody against the B-cell specific CD20-antigen, has been demonstrated to be effective in the treatment of non-Hodgkin's B-cell lymphoma (B-NHL). Previous in vitro studies have shown that direct complement-dependent cytotoxicity (CDC), ADCC and apoptosis are important in the rituximab-induced killing of lymphoma cells. It is, however, unknown whether rituximab penetrates the blood-brain barrier. Therefore, we studied rituximab levels and complement (C) activation in blood and cerebrospinal fluid (CSF) following intravenous rituximab therapy in a patient with relapsing non-Hodgkin's lymphoma with central nervous system (CNS) involvement. Longitudinal samples from blood and CSF were taken at 13 time-points during the treatment period. The results show that the C cascade becomes activated in blood during the first mAb infusion (C3a-desArg concentration rose from 55 to 138 microg/ml during the first 2 hours). After the first infusion the proportions of lymphocytes positive for the CD19- and CD20-antigens in the peripheral blood were reduced from 41% and 35%, respectively, to a level of 2% (for both). In CSF the rituximab concentration increased after successive infusions, but remained below 0.55 microg/ml (compared to a Cmax of 400 microg/ml in peripheral blood). Although a minor and delayed C activation response was seen in the CSF the treatment did not clear CD20-positive cells away from the CNS. Thus, it appears that an intact blood-brain barrier restricts the entry of rituximab into the CNS. Possible options to circumvent this would be dose escalation or intrathecal rituximab treatment.

Summary Background Chemotherapy drug distribution varies greatly among individual patients. Therefore, we developed an individualised fluorouracil, epirubicin, cyclophosphamide (FEC) regimen to improve outcomes in patients with high-risk early breast cancer. We then did a randomised trial to compare this individually tailored FEC regimen with conventional adjuvant chemotherapy followed by consolidation with high-dose chemotherapy with stem-cell support. Methods 525 women younger than 60 years of age with highrisk primary breast cancer were randomised after surgery to receive nine cycles of tailored FEC to haematological equitoxicity with granulocyte colony-stimulating factor (G-CSF) support (n=251), or three cycles of FEC at standard doses followed by high-dose chemotherapy with cyclophosphamide, thiotepa, and carboplatin (CTCb), and peripheral-blood stemcell or bone-marrow support (n=274). Both groups received locoregional radiation therapy and tamoxifen for 5 years. The primary outcome measure was relapse-free survival, and analysis was by intention to treat. Findings At a median follow-up of 34·3 months, there were 81 breast-cancer relapses in the tailored FEC group versus 113 in the CTCb group (double triangular method p=0·04). 60 deaths occurred in the tailored FEC group and 82 in the CTCb group (log-rank p=0·12). Patients in the CTCb group experienced more grade 3 or 4 acute toxicity compared with the tailored FEC group (p Interpretation Tailored FEC with G-CSF support resulted in a significantly improved relapse-free survival and fewer grade 3 and 4 toxicities compared with marrow-supported high-dose chemotherapy with CTCb as adjuvant therapy of women with high-risk primary breast cancer.

Background: Men with testicular cancer are at an increased risk of leukemia, but the relationship to prior treatments is not well characterized. The purpose of our study was to describe the risk of leukemia following radiotherapy and chemotherapy for testicular cancer. Methods: Within a population-based cohort of 18 567 patients diagnosed with testicular cancer (from 1970 through 1993), a case-control study of leukemia was undertaken, Radiation dose to active bone marrow and type and cumulative amount of cytotoxic drugs were compared between 36 men who developed leukemia and 106 matched control patients without leukemia. Conditional logistic regression was used to estimate the relative risk of leukemia associated with specific treatments. All P values are two-sided. Results: Radiotherapy (mean dose to active bone marrow, 12.6 Gy) without chemotherapy was associated with a threefold elevated risk of leukemia. Risk increased with increasing dose of radiation to active bone marrow (P for trend = .02), with patients receiving radiotherapy to the chest as well as to the abdominal/pelvic fields accounting for much of the risk at higher doses. Radiation dose to active bone marrow and the cumulative dose of cisplatin (P for trend = .001) were both predictive of excess leukemia risk in a model adjusted for all treatment variables. The estimated relative risk of leukemia at a cumulative dose of 650 mg cisplatin, which is commonly administered in current testicular cancer treatment regimens, was 3.2 (95% confidence interval = 1.5-8.4); larger doses (1000 mg) were linked with statistically significant sixfold increased risks. Conclusions: Past treatments for testicular cancer are associated with an increased risk of leukemia, with evidence for dose-response relationships for both radiotherapy and cisplatin-based chemotherapy. Statistically nonsignificant excesses are estimated for current radiotherapy regimens limited to the abdomen and pelvis: Among 10 000 patients given a treatment dose of 25 Gy and followed for 15 years, an excess of nine leukemias is predicted; cisplatin-based chemotherapy (dose, 650 mg) might result in 16 cases of leukemia. The survival advantage provided by current radiotherapy and chemotherapy regimens for testicular cancer far exceeds the small absolute risk of leukemia.

PURPOSE: To quantify the risk of second cancers among long-term survivors of Hodgkin’s disease (HD) diagnosed before 21 years of age and to explore sex-, age-, and site-related differences. PATIENTS AND METHODS: We analyzed data from 5,925 pediatric HD patients, including 2,646 10-year and 755 20-year survivors, who were reported to 16 population-based cancer registries in North America and Europe between 1935 and 1994. RESULTS: A total of 157 solid tumors (observed/expected ratio [O/E] = 7.0; 95% confidence interval [CI], 5.9 to 8.2.) and 26 acute leukemias (O/E = 27.4; 95% CI, 17.9 to 40.2) were reported. Risk of solid tumors remained significantly increased among 20-year survivors (O/E = 6.6, observed [O] = 40, cumulative risk = 6.5%) and persisted for 25 years (O/E = 4.6, O = 15, cumulative risk = 11.7%). Temporal trends for cancers of thyroid, female breast, bone/connective tissue, stomach, and esophagus were consistent with the late effects of radiotherapy. Greater than 50-fold increased risks were observed for tumors of the thyroid and respiratory tract (one lung and one pleura) among children treated before age 10. At older ages (10 to 16 years), the largest number of second cancers occurred in the digestive tract (O/E = 19.3) and breast (O/E = 22.9). Risk of solid tumors increased with decreasing age at HD on a relative but not absolute scale. CONCLUSION: Children and adolescents treated for HD experience significantly increased risks of second cancers at various sites for 2 to 3 decades. Although our results reflect the late effects of past therapeutic modalities, they underscore the importance of lifelong follow-up of pediatric HD patients given early, more aggressive treatments.

A small but not insignificant number of patients experience a prolonged survival after treatment of metastatic soft tissue sarcoma. This must be weighed against the majority of the patients who benefit little from the therapy, but nevertheless experience its side-effects. It would therefore be of utmost importance to be able to screen for those patients who respond to the treatment. Since proliferating cells are more sensitive to chemotherapy than non-proliferative cells, we measured the proliferation rate of the primary tumour of 55 soft tissue sarcoma patients with locally advanced or metastatic disease by determining the flow cytometric S phase fraction and immunohistochemical Ki-67 and cyclin A scores. S phase fraction or Ki-67 score did not predict chemotherapy response or progression-free survival. A high cyclin A score, however, correlated with a better chemotherapy response (P = 0.02) and longer progression-free survival time (P = 0.04). Our results suggest that a high cyclin A score predicts chemotherapy sensitivity. © 1999 Cancer Research Campaign

The aims of the present study were to compare genetic aberrations in primary sarcomas and their pulmonary metastases and to explore the pathways associated with disease spreading. The primary tumor and its subsequent pulmonary metastasis of 22 patients were analyzed by comparative genomic hybridization. All samples were obtained before the initiation of chemo- or radiotherapy. The mean total number of aberrations per tumor was 7.6 (range, 0–17) in primary tumors and 7.5 (range, 0–19) in metastases. The mean numbers of high-level amplifications per tumor were similar (0.32 in primary tumors and 0.36 in metastases). The frequencies of the most common aberrations were relatively similar in primary tumors and metastases: the most frequent gain affected 1q (minimal common regions 1q21–q23 in 36% of primary tumors and 1q21 in 45% of metastases). The most frequent losses were detected at 9p (9p22–pter in 32% of primary tumors and 9p21–pter in 32% of metastases), 10p (10p11.2–p12 in 41% of primary tumors and 10p11.2–pter in 32% of metastases), 11q (11q23–qter in 36% of primary tumors and 32% of metastases), and 13q (13q14–q21 in 45% of primary tumors and 50% of metastases). No aberrations specific to metastases were detected. An increase in the total number of changes during progression was a predominant feature in a majority of these paired samples. Also, the number of differences in the genetic profile outnumbered common changes in a majority of the samples. However, despite the heterogeneous and numerous changes, all pairs with aberrations in both specimens had some shared alterations in both samples. Genes Chromosomes Cancer 25:323–331, 1999. © 1999 Wiley-Liss, Inc.

Immunohistochemically determined Ki-67 scores and flow cytometrically determined S-phase fractions were successfully evaluated from the primary tumours of 123 patients with soft-tissue sarcoma. All patients had either limb or superficial trunk tumours. Ki-67 score correlated strongly with ploidy, S-phase fraction and grade. Ki-67 did not correlate with the size of the primary tumour. When analysed as a continuous variable, Ki-67 was a stronger predictor of both metastasis-free survival and disease-specific overall survival (P= 0.003 and 0.04 respectively) than was the S-phase fraction (P= 0.06 and 0.07 respectively). We tested the relevance of different cut-point values by dividing the whole material into two parts at every 10% (e.g. 10% of patients vs. the remaining 90%, 20% vs. 80%, etc.). We counted the relative risk and confidence interval at all these cut-off points. Ki-67 had good prognostic discriminating power irrespective of the cut-point value, but S-phase fraction lost its prognostic power at higher cut-point values. In conclusion, we found that Ki-67 is a useful prognostic tool in the treatment of soft-tissue sarcoma patients irrespective of the cut-point value. S-phase fraction can be used at lower cut-point values. © 1999 Cancer Research Campaign

The first chemotherapy study of soft tissue sarcoma (STS) by the Scandinavian Sarcoma Group was started in 1981 (SSG I). It evaluated the single agent adjuvant doxorubicin in a randomized setting in patients with high-grade STS. No improvement was noted in the overall survival or disease-free survival rate. More intense chemotherapy was thereafter (1991-1994) evaluated in a phase 2 study, introducing ifosfamide and a continuous infusion of etoposide with growth factor (SSG X). The response rate of previously untreated patients was high (42%), but complete remissions were few. Analysis of patients undergoing surgery after preoperative chemotherapy suggested an increased survival. A recent meta-analysis of adjuvant chemotherapy for localized resectable STS in adults, including the SSG I trial, indicated a better disease-free survival and possibly improved overall survival (Thierny et al. 1997). At present, we are studying whether such a benefit can be shown in patients with high-risk prognostic criteria by giving adjuvant ifosfamide and doxorubicin treatment after primary surgery (SSG XIII). In the latter SSG study, started on July 1, 1998, the adjuvant therapy is evaluated in a phase 2 study in selected patients with high-grade STS and other unfavorable prognostic factors.

The feasibility of mobilizing peripheral blood stem cells (PBSC) using anthracycline containing polychemotherapy and G-CSF on the first 50 patients randomized to the high-dose arm in the adjuvant SBG 9401 is investigated. The patients were treated with standard FEC (5-fluorouracil 600 mg/m2, epirubicin 60 mg/m2, cyclophosphamide 600 mg/m2) for two courses followed by a modified third FEC course with a C dose of 1200 mg/m2 supported with subcutaneous G-CSF (filgrastim) at 5 mg/kg followed by harvest around day 11. The mean yield of CD34+

(1999). Chemotherapy in osteosarcoma. Acta Orthopaedica Scandinavica: Vol. 70, The Scandinavian Sarcoma Group: Twenty years' experience, pp. 74-82.

Purpose: To study the feasibility of a dose-escalated accelerated hyperfractionation schedule for patients with advanced head and neck cancer. Materials and Methods: Twenty-nine previously untreated patients with advanced squamous cell carcinoma were treated with the following biweekly dose-escalated accelerated hyperfraction schedule: during the first 2 weeks 1.2 Gy twice a daily (bid) up to 24 Gy, thereafter during the next following 2 weeks 1.4 Gy bid to 28 Gy in 20 fractions, and thereafter 22.4 Gy in 1.6 Gy bid fractions during 11/2 weeks. Thus, the the total dose was 74.4 Gy in 54 fractions given in 51/2 weeks, Results: The planned total dose was given within the planned time to 19 (66%) patients. For seven patients the treatment time was prolonged with 1 to 6 days because of department closure for holidays or machine-down days, and in three cases the treatment time was prolonged more than 8 weeks. When the tumor responses were evaluated at 3 months after given radiotherapy, 27 (93%) patients showed complete tumor clearance, 1 patient had a recidual focus, and 1 patient showed progressive disease. The ultimate 1-, 2-, and 3-year local control rates were: 87, 71, and 60%. Four patients had a salvage laryngectomy. The 1-, 2-, and 3-year survival rates for all patients were as follows: 96, 81, and 73%. All patients developed confluent mucositis, 15 patients were hospitalized for nutritional support, and 11 patients had moist desquamation. However, all acute reactions healed completely, and no serious late complications were observed. Conclusions: This is a safe and effective treatment schedule for patients with advanced head and neck cancer.

The intensified induction regimens used and the potential use of high-dose consolidation chemotherapy (CT) in advanced soft tissue sarcomas (STS) has focused interest on the outcome of those patients who can achieve complete remission (CR) by current therapy. The files from four institutions with a special interest in STS were studied. 38 adult patients with advanced STS who were converted disease-free by either CT alone (n = 14) or CT followed by surgery (n = 24) were found. The median follow-up time was 29 months. The median disease-free survival (DFS) was 18 months and the estimated 2-year DFS 34%. The median disease-specific survival (DSS) was 40 months and the estimated 2-year DSS 78%. For patients who achieved CR by CT alone, and for patients who were converted to CR by surgery, the corresponding DFS figures were 23 months (estimated 2 year DFS 48%) and 10 months (26%) (P = 0.07), respectively. The histological response to CT significantly predicted outcome in patients subjected to surgery (DFS P value 0.004, DSS P value 0.02). Patients who achieved CR by surgery shortly after having achieved a clinical partial response (PR with early surgery) did better than those who where converted to CR by surgery after protracted CT following a clinical PR (PR with late surgery) (DFS P value 0.02, DSS P value 0.1). Our results confirm that CT alone can induce prolonged DFS in rare patients with advanced STS. In patients subjected to surgery, a good histological response indicates improved outcome.

Background Adjuvant chemotherapy has improved the outcome of childhood malignant brain tumors in large randomized trials. With increasing survival rates, treatment toxicity has become a matter of concern. Radiation therapy and cisplatinum are known to be ototoxic. Methods We evaluated the incidence and factors predisposing to ototoxicity in children treated with the “8 in 1” chemotherapy protocol in Finland during 1986–1993. Thirty-five of the 82 children survived for at least 1 year after diagnosis. Thirty of these children were old enough to have a audiogram. Results Seventeen of the 30 children had normal hearing, seven had hearing loss at high frequencies, and six (20%) had severe hearing loss in the speech range. The risk factors for severe hearing loss were young age, a high cumulative dose of cisplatinum, and deteriorating renal function. In the presence of these factors, the risk of severe hearing loss was over 50%. Hearing loss at high frequencies could occur after low cumulative doses of cisplatinum, but severe hearing loss correlated with high cumulative doses. Conclusions Cisplatinum-induced hearing loss at high frequencies is common, but hearing loss in the speech range also occurs, particularly in children with predisposing factors, and may progress insidiously and rapidly. Therefore a hearing test before each “8 in 1” course is important. © 1996 Wiley-Liss, Inc.

A significant correlation (r = -0.48) was found between the logarithm of the S-phase fraction of the primary tumour (SPF) and the logarithm of the doubling time of lung metastases (T2). The estimated median cell loss factor was 88% (range 35-99%).

Three biochemical markers of collagen metabolism were measured in 39 osteosarcoma patients. The pretreatment values did not predict outcome, and the markers showed no consistent change upon development of metastases. Both the age of the patients and the multimodality therapy affected the collagen metabolites. These findings emphasise the need for cautious interpretation of tumour-associated markers.

We report the results of cytogenetic analysis of malignant fibrous histiocytoma of soft tissue (MFH). Seven of 12 successfully cultured MFHs had complex clonal aberrations, including translocations, deletions, and unidentifiable marker chromosomes. Telomeric associations were observed in five and the double minute phenomenon in four of seven MFHs with abnormal karyotypes. In one case (a storiform-pleomorphic MFH, grade IV) with a complex polyploid karyotype, two clonal ring chromosomes were present, one interpreted as r(19)(p13q13), one unidentified. In two tumors, clonal structural rearrangements of chromosome 1 were seen: del(1)(q21) in a storiform-pleomorphic MFH, grade IV, and add(1)(q21 or q32),t(1;10)(p22;q22) in a myxoid MFH, grade I. The remaining five MFHs had normal karyotypes, but in two of them nonclonal, structural aberrations were found. The modal chromosome number in the studied MFHs varied widely, but the majority of tumors with abnormal karyotypes had polyploid chromosome complements (five of seven cases). Our results confirm many of the previous findings and indicate that double minutes (dmins) may be more frequent in MFH than previously reported.

This study was conducted to determine antibiotic susceptibility of Flavobacterium psychrophilumisolated from diseased rainbow trout (Oncorhynchus mykiss)from two hatcheries in the Mediterranean region of Turkey. Tissue samples for bacteriology were taken from rainbow trout fry (body weight 1-2g) in April 2015. The samples were collected from kidney and spleen and streaked onto tryptone yeast extract salts (TYES) agar. Agar plates were incubated at 15C for 72 hours. Fourteen isolates were identified as F. psychrophilum using biochemical, physiological and morphological characteristics and API ZYM test. Antibiotic sensitivities of the isolates were determined by the disc diffusion method on TYES agar. The results were evaluated according to NCCLS standards. Only one isolate from each hatchery was used for antibiogram test. Both of them were sensitive to amoxicillin/clavulanic acid 2:1, penicillin, nitrofurantoin, florfenicol, amoxicillin, clindamycin, chloramphenicol, ampicillin, doxycycline, streptomycin, and erythromycin. The tested isolates were resistant to sulfadiazine, sulfamethoxazole/trimethoprim, oxolinic acid, vancomycin, trimethoprim/sulfadiazine, gentamicin, kanamycin, nalidixic acid, oxacillin, enrofloxacin, flumequine, tobramycin. While the examined isolates were sensitive to florfenicol, they were resistant to sulfonamides, which is in general agreement with antibiotic sensitivity studies of F. psychrophilum from other geographical regions. The results indicated that antimicrobial susceptibility testing of F. psychrophilum from each region is important issue to be able to combat disease.

Background The aim was to evaluate the effect of a 12-month individualized health coaching intervention by telephony on clinical outcomes. Methods An open-label cluster-randomized parallel groups trial. Pre- and post-intervention anthropometric and blood pressure measurements by trained nurses, laboratory measures from electronic medical records (EMR). A total of 2594 patients filling inclusion criteria (age 45 years or older, with type 2 diabetes, coronary artery disease or congestive heart failure, and unmet treatment goals) were identified from EMRs, and 1535 patients (59%) gave consent and were randomized into intervention or control arm. Final analysis included 1221 (80%) participants with data on primary end-points both at entry and at end. Primary outcomes were systolic and diastolic blood pressure, serum total and LDL cholesterol concentration, waist circumference for all patients, glycated hemoglobin (HbA1c) for diabetics and NYHA class in patients with congestive heart failure. The target effect was defined as a 10-percentage point increase in the proportion of patients reaching the treatment goal in the intervention arm. Results The proportion of patients with diastolic blood pressure initially above the target level decreasing to 85 mmHg or lower was 48% in the intervention arm and 37% in the control arm (difference 10.8%, 95% confidence interval 1.5–19.7%). No significant differences emerged between the arms in the other primary end-points. However, the target levels of systolic blood pressure and waist circumference were reached non-significantly more frequently in the intervention arm. Conclusions Individualized health coaching by telephony, as implemented in the trial was unable to achieve majority of the disease management clinical measures. To provide substantial benefits, interventions may need to be more intensive, target specific sub-groups, and/or to be fully integrated into local health care. Trial registration ClinicalTrials.gov Identifier: NCT00552903

We have previously shown that the serum aminoterminal propeptide of type III procollagen (PIIINP) is a prognostic factor for survival in localised soft-tissue sarcomas, and that elevated values are frequent in metastatic disease. In the present study PIINP is analysed during chemotherapy in 26 patients with advanced sarcomas. Non-responders had a significantly higher pretreatment level of PIIINP than responders (P = 0.05), when only patients with no recent therapeutic interventions were studied. However, during chemotherapy PIIINP followed the clinical course of the malignant disease in only a minority of patients. Patients with recent surgery or recently completed chemotherapy had an increased pretreatment PIIINP value (P = 0.03). In these patients PIIINP declined during chemotherapy irrespective of tumour response. A pretreatment PIIINP level within the reference range tended to increase with time irrespective of response. Moreover, the values taken during a chemotherapy infusion were significantly higher than those immediately preceding the corresponding cycle (P = 0.001). Our results suggest that pretreatment PIIINP is of value as a prognostic factor for chemotherapy response in patients with advanced sarcomas. During chemotherapy PIIINP is of minor importance in monitoring response because of the influence of chemotherapy and other therapeutic interventions on the level of PIIINP.

In a randomised Scandinavian Sarcoma Group study (n = 240) on the effect of postoperative adjuvant doxorubicin in high grade adult soft tissue sarcoma, 26 patients were treated with marginal surgery and postoperative radiotherapy. The protocol dose was 51 Gy in 17 fractions, or equivalent. Local recurrence occurred in 6 patients. Two local failures were geographical misses. Salvage treatment was ultimately successful in 3 of 4 attempted cases. 15 patients had complications, which in 3 cases necessitated amputation. These 3 patients had received the protocol fractionation and doxorubicin. However, other factors possibly responsible for the complication were also present.

The volume doubling time (T2) of 52 lung metastases in 21 patients was calculated from measurements done on plain chest radiographs. Follow-up times ranged from 14 to 819 days. The measurements were fairly well reproducible in the majority of patients, although considerable discrepancies in T2 estimates made by two independent observers were found in a few patients. The median doubling time was 34.9 days (estimated 95% range 3.9 to 352 days). The variation of T2:s between patients was significantly (P = 0.0001) larger than that between T2: of multiple metastases in the same patients. The growth of the metastases seemed to be well described by a simple exponential function in all patients with more than two measurements, without evidence of Gompertzian growth. There seemed to be a linear correlation between the logarithm of T2 and log-survival time from diagnosis of metastatic disease, even if only one third of the variation of survival times between patients could be explained by differences in T2. T2 was not a significant factor for survival in Cox-analysis (P = 0.10).

The hemolytic activity of cells of smooth and rough phenotypic variants of the Gram-negative fish pathogen Flavobacterium psychrophilum was investigated in two different assays, a microplate and an agarose hemolysis assay, using rainbow trout erythrocytes. The smooth cells showed a high and the rough cells a negligible, concentration dependent, hemolytic activity in the microplate assay. Both smooth and rough cells showed a rather weak hemolytic activity, with two distinct hemolytic patterns, in the agarose assay. The hemolytic activity of the cells was not regulated by iron availability and cell-free extracellular products did not show any hemolytic activity. The smooth cells, in contrast to the rough cells, showed a high ability to agglutinate erythrocytes and both hemagglutination and hemolytic activity was impaired by treatment of the cells with sialic acid. The hemolytic activity was furthermore reduced after proteolytic and heat treatment of the cells. The results from the present study suggest that the hemolytic activity in F. psychrophilum is highly expressed in the smooth phenotype, and that it is a contact-dependent and two-step mechanism that is initiated by the binding of the bacterial cells to the erythrocytes through sialic acid-binding lectins and then executed by thermolabile proteinaceous hemolysins.

Thirty-three cases of postirradiation sarcoma (PIS) from the files of the Finnish Cancer Registry were analyzed. The most frequent first primary tumors were cancers of the breast (seven cases) and female reproductive organs (13 cases). Five patients had a childhood cancer. The median total radiation dose at the site of the PIS was 3600 cGy (1600 cGy to 11200 cGy). The median interval from start of radiation therapy to detection of PIS was 13.2 years (3.4 to 22.8 years). The PIS was of soft tissue origin in 25 of 33 cases. The most frequent histologic types were osteosarcoma (ten cases, including four extraskeletal tumors), malignant fibrous histiocytoma (ten cases), and fibrosarcoma (six cases). The overall crude 5-year survival rate was 29% (calculated from the start of treatment for PIS), and for patients initially treated with either radical surgery or combined marginal surgery and postoperative irradiation it was 67%. The authors conclude that there is a chance for cure for radically treated patients with postirradiation sarcoma that emphasizes the importance of regular long-term follow-up of cancer patients.

Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome has become a widespread fish pathogen in freshwater aquaculture worldwide. In this study, a low molecular mass fraction (P25-33), with an approximate weight of 25-33 kDa, was identified among F. psychrophilum strains in an immunoblotting analysis with anti-F. psychrophilum sera. The immunogenic efficacy of the isolated and extracted P25-33 was investigated in two intraperitoneal immunization trials with rainbow trout, Oncorhynchus mykiss (Walbaum). The first trial included immunizations using P25-33 with Freund's complete adjuvant (FCA) and the second trial included immunizations using P25-33, formalin-inactivated whole and sonicated F. psychrophilum cell preparations without FCA. In both trials, antibody titres against F. psychrophilum were analysed with an enzyme-linked immunosorbent assay and the efficacy of the immunizations was determined by a challenge with F. psychrophilum. The P25-33 was shown to give rise to a protective immune response in rainbow trout after immunization with FCA, but not without FCA when a low concentration of P25-33 was used. Instead formalin-inactivated whole and sonicated cells of F. psychrophilum were able to protect the immunized fish more effectively when immunized without FCA. The results suggest that whole or sonicated F. psychrophilum cells could be better candidates for a cost-effective water-based injection vaccine than the immunogenic fraction.

Endocrine disrupting compounds are chemicals that may interfere with the endocrine system causing severe effects in organisms. The three-spined stickleback (Gasterosteus aculeatus L.) offers a potential for the assessment of endocrine disruption caused by a) estrogenic xenobiotics through the estrogen-dependent protein vitellogenin and b) androgenic xenobiotics through the androgen-dependent protein spiggin. The stickleback is presently the only known fish species with a quantifiable androgen and anti-androgen biomarker endpoint. In the current study, hepatocyte and kidney primary cell cultures and liver and kidney tissue slice cultures were prepared and used for detecting estrogenic or androgenic activity in vitro through the action of hormones or municipal sewage water. The results indicate that stickleback male hepatocyte cultures are suitable in detecting estrogenic activity and stickleback female kidney tissue slice cultures in detecting androgenic activity. The tested sewage water showed high estrogenic activity but no significant androgenic activity. Primary cell and tissue slice cultures isolated from the three-spined stickleback will allow simultaneously screening in vitro for potential estrogenic and androgenic activity of complex samples.

Highly emetogenic drugs such as cisplatin induce an increase in the urinary 5-hydroxyindoleacetic acid (5-HIAA) level, the main metabolite of serotonin (5-HT), within the first 24 h following a single infusion, thus providing a possible cause for acute emesis and an explanation for the action of 5-HT3 antagonists. No further excretion peaks have been observed, suggesting that additional or serotonin-independent mechanisms cause delayed emesis. Our aim was to study the mechanisms behind emesis seen during a highly emetogenic chemotherapy regimen given as a continuous infusion over several days. Seven women treated with a 4-day high-dose chemotherapy (HDCT) regimen for breast cancer entered the study. Pooled urine samples were collected prior to and during chemotherapy for determining 5-HIAA excretion. An excretion peak in the urinary 5-HIAA level was observed within the first 24 h with no further peaks thereafter. Thus, the mechanisms behind the emesis experienced during this highly emetogenic multiple-day chemotherapy regimen from days 2-3 onwards would appear to be at least partially serotonin independent and would not be expected to be completely relieved by 5-HT3 antagonists alone.

Purpose: Breast carcinomas expressing basal epithelium cytokeratins constitute a tumor subgroup that is typically hormone receptor negative and shows a distinct gene expression profile. Based on variable basal cytokeratin immunostaining patterns, we hypothesized that the “basal phenotype” tumor group may comprise more than one biological entity. Experimental Design: Basal cytokeratins 5 and 14 (CK5/14) were stained by immunohistochemistry and the percentage of positive cells was defined by image analysis. The results thus obtained were compared with clinicopathologic characteristics and relapse-free survival. Results: Of the 506 breast tumors, 53 (10.5%) showed immunoreactivity for CK5/14. Basal cytokeratin expression showed up as two microscopically distinguishable subtypes, i.e., a uniformly positive type (“basal”) and a partially positive type (“basoluminal”) often displaying a checkerboard-type intratumoral heterogeneity. These subgroups could also be separated with a third basal cytokeratin (CK17, P < 0.0001). Both basal and basoluminal subtypes were hormone receptor negative and of high grade, but differed with respect to the Ki-67 labeling index (P = 0.0014), vimentin (P = 0.005), and c-kit (P = 0.02), which were more frequently expressed in basal than in basoluminal tumors. In contrast, the amplification of HER-2 was found almost exclusively in the basoluminal subgroup (P = 0.009). Compared with the basal tumors, basoluminal tumors associated with significantly shorter relapse-free survival (P = 0.01), which was not explained by their more frequent HER-2 amplification. Conclusions: We conclude that the intratumoral heterogeneity in basal cytokeratin expression can be used to define two distinct breast cancer subtypes, basal and basoluminal, with distinctive features related to proliferation activity, oncogene and biomarker status, and patient survival.

Women with stage III ovarian cancer and with < or = 2 cm residual tumour were randomly assigned to receive either conventionally dosed chemotherapy (group A) or HDCT (group B). Patients allocated to group A received 6 cycles of paclitaxel (T) 135 mg/m2 and cisplatin (P) 75 mg/m2 every 3 weeks, and those allocated to HDCT received 3 TP cycles followed by peripheral blood stem cell mobilisation with cyclophosphamide (C) 3000 mg/m2 and T 175 mg/m2, and subsequently HDCT with carboplatin 1500 mg/m2, C 120 mg/kg, and mitoxantrone 75 mg/m2. The trial was closed early after 42 patients were entered due to slow accrual. The median follow-up time of patients who were alive was 81 months. The median progression-free survival time was 15.9 and 16.6 months (hazard ratio, HR 0.83; 95% CI 0.41-1.69, P = 0.61) and the median overall survival time was 43.7 and 64.3 months (HR, 0.74; 95% CI 0.34-1.61, P = 0.44) in groups A and B, respectively. Although one patient died of HDCT-related toxicity, the regimen was otherwise relatively well tolerated. We conclude that the HDCT regimen used was feasible, but did not result in significantly improved survival in this prematurely closed trial. A clinically important survival benefit cannot be excluded due to the small sample size.