PRECISION: the Belgian molecular profiling program of metastatic cancer for clinical decision and treatment assignment.

PRECISION is an initiative from the Belgian Society of Medical Oncology (BSMO) in collaboration with several stakeholders, encompassing four programs that aim to boost genomic and clinical knowledge with the ultimate goal to offer patients with metastatic solid tumors molecularly guided treatments. The PRECISION 1 study has led to the creation of a clinico-genomic database. The Belgian Approach for Local Laboratory Extensive Tumor Testing (BALLETT) and GeNeo studies will increase the number of patients with advanced cancer that have comprehensive genotyping of their cancer. The PRECISION 2 project consists of investigator-initiated phase II studies aiming to provide access to a targeted drug for patients whose tumors harbor actionable mutations in case the matched drug is not available through reimbursement or clinical trials in Belgium.

Characterization of male breast cancer: results of the EORTC 10085/TBCRC/BIG/NABCG International Male Breast Cancer Program.

Background: Male breast cancer (BC) is rare, managed by extrapolation from female BC. The International Male BC Program aims to better characterize and manage this disease. We report the results of part I, a retrospective joint analysis of cases diagnosed during a 20-year period. Methods: Patients with follow-up and tumor samples, treated between 1990 and 2010, in 93 centers/9 countries. Samples were centrally analyzed in three laboratories (the United Kingdom, the Netherlands and the United States). Results: Of 1822 patients enrolled, 1483 were analyzed; 63.5% were diagnosed between 2001 and 2010, 57 (5.1%) had metastatic disease (M1). Median age at diagnosis: 68.4 years. Of 1054 M0 cases, 56.2% were node-negative (N0) and 48.5% had T1 tumors; 4% had breast conserving surgery (BCS), 18% sentinel lymph-node biopsy; half received adjuvant radiotherapy; 29.8% (neo)adjuvant chemotherapy and 76.8% adjuvant endocrine therapy (ET), mostly tamoxifen (88.4%). Per central pathology, for M0 tumors: 84.8% ductal invasive carcinomas, 51.5% grade 2; 99.3% estrogen receptor (ER)-positive; 81.9% progesterone receptor (PR)-positive; 96.9% androgen receptor (AR)-positive [ER, PR or AR Allred score ≥3]; 61.1% Ki67 expression low (<14% positive cells); using immunohistochemistry (IHC) surrogates, 41.9% were Luminal-A-like, 48.6% Luminal-B-like/HER-2-negative, 8.7% HER-2-positive, 0.3% triple negative. Median follow-up: 8.2 years (0.0-23.8) for all, 7.2 years (0.0-23.2), for M0, 2.6 years (0.0-12.7) for M1 patients. A significant improvement over time was observed in age-corrected BC mortality. BC-specific-mortality was higher for men younger than 50 years. Better overall (OS) and recurrence-free survival (RFS) were observed for highly ER+ (P = 0.001), highly PR+ (P = 0.002), highly AR+ disease (P = 0.019). There was no association between OS/RFS and HER-2 status, Ki67, IHC subtypes nor grade. Conclusions: Male BC is usually ER, PR and AR-positive, Luminal B-like/HER2-negative. Of note, 56% patients had T1 tumors but only 4% had BCS. ER was highly positive in >90% of cases but only 77% received adjuvant ET. ER, PR and AR were associated with OS and RFS, whereas grade, Ki67 and IHC surrogates were not. Significant improvement in survival over time was observed.

Autonomic Dysfunction Precedes Development of Rheumatoid Arthritis: A Prospective Cohort Study.

BACKGROUND: Heart rate variability (HRV) is a validated method to establish autonomic nervous system (ANS) activity. Rheumatoid arthritis (RA) is accompanied by ANS imbalance. We hypothesized that ANS dysfunction may precede the development of RA, which would suggest that it plays a role in its etiopathogenesis. METHODS: First, we assessed HRV parameters in supine (resting) and upright (active) position in healthy subjects (HS, n=20), individuals at risk of developing arthritis (AR subjects, n=50) and RA patients (RA, n=20). Next, we measured resting heart rate (RHR), a parasympathetic HRV parameter, in an independent prospective cohort of AR subjects (n=45). We also evaluated expression levels of the parasympathetic nicotinic acetylcholine receptor type 7 (α7nAChR) on circulating monocytes. FINDINGS: Both AR subjects (68 beats per minute (bpm), interquartile range (IQR) 68-73) and RA patients (68bpm, IQR 62-76) had a significantly higher RHR compared to HS (60bpm, IQR 56-63). RHR was significantly higher at baseline in individuals who subsequently developed arthritis. Expression levels of α7nAChR were lower in AR subjects with RHR ≥70bpm compared to those with RHR <70bpm, consistent with reduced activity of the parasympathetic cholinergic anti-inflammatory pathway. INTERPRETATION: These data support the notion that autonomic dysfunction precedes the development of RA.

Parkinson's disease, levodopa-use and the risk of melanoma.

Since the early 1970s, the literature has suggested an association between Parkinson's Disease (PD) and/or levodopa-use and an increased risk for the development of malignant melanoma. In some countries, this possible association has even led to a warning in the drug insert leaflet of the possible risk. Recently, five studies have been published that have investigated both associations and three conclusions can be drawn. Firstly, there appears to be an increased risk in the development of melanomas in patients with PD. Secondly, this increased risk is already present before the PD is diagnosed. Finally, it is unlikely that levodopa plays any role in this phenomenon. It is not known which factors are responsible for this increase in the development of melanomas in PD patients and this needs further investigation. We recommend the removal of the warning from the drug insert leaflet, since this can lead to unnecessary fear on the part of the patients and physician resistance to prescribing this medication.

Transduction of ovarian cancer cells: a recombinant adeno-associated viral vector compared to an adenoviral vector.

Recombinant adeno-associated virus (rAAV) vectors have emerged as vehicles for gene therapy. In addition, anti-neoplastic properties have been attributed to wild-type AAV. To take advantage of both features and to overcome technical problems associated with rAAV preparation, we developed a production method in which rAAV particles are amplified in an infectious cycle in the presence of wtAAV. This results in a 10(3)-10(4)-fold amplification of rAAV input particles. rAAV-GFP particles generated by this method were used to transduce ovarian cancer cell lines to evaluate their potential in ovarian cancer gene therapy, in comparison to a rAd-GFP vector. The transduction efficiency of NIH-OVCAR3, MDAH 2774 and SKOV3 cells with rAAV-GFP particles was low (< 1%) and did not improve by increasing the number of particles/cell. Repeated administration and continued exposure of NIH-OVCAR3 and MDAH 2774 improved transduction to over 3%. In contrast, these cell lines were more efficiently transduced by rAAV-GFP in the presence of adenovirus (approximately 15%) and by rAd-GFP (> 50%). These results indicate that in contrast to rAd vectors, rAAV particles are not suitable for therapeutic gene transfer in ovarian cancer cells unless efficient help can be provided to mediate ss to ds DNA conversion.

Contaminants within bacterial plasmid preparations trigger apoptosis in liposome transfected OVCAR3, but not in SKOV3 or AZ224 human ovarian cancer cells.

Toxicity associated with plasmid/liposome transfection of eucaryote cells has been attributed to the inherent toxicity of cationic lipid formulations and also to bacterial contaminants of plasmid DNA preparations, such as lipopolysaccharides (LPS). Certain plasmid preparations were observed to trigger apoptosis in DNA/liposome transfected OVCAR3 human epithelial ovarian cancer cells. In contrast, AZ224 and SKOV3 cells were unaffected under the same conditions. Agarose gel electrophoresis with recovery of the plasmid DNA removed the toxic component, but not purification by phenol/chloroform extraction or isopicnic CsCl ultracentrifugation. The toxicity of individual preparations correlated with the concentration of bacterial LPS. However, polymixin B could not neutralise the toxicity and neither could the effect be reproduced by the addition of bacterial LPS to non-toxic plasmid preparations. Surprisingly, the conditioned medium of OVCAR3 cells undergoing apoptosis was found to kill non-transfected OVCAR3 cells but not AZ224 or SKOV3 cells. This observation illustrates the possibility that unpredictable contaminants of bacterial plasmid preparations are able to cause cell death in the context of plasmid/liposome transfection in a cell-type specific way. It emphasizes the importance of achieving maximal plasmid DNA purity when performing DNA transfection experiments that focus on cell survival.

Characterization of permanent cell lines that contain the AAV2 rep-cap genes on an Epstein-Barr-virus-based episomal plasmid.

Recombinant adeno-associated virus (rAAV) has emerged as a promising gene therapy vector. Its development, however, has been hampered by the lack of a readily available efficient production method. We investigated the possibility of establishing permanent cell lines for the production of rAAV with a new Epstein-Barr-virus (EBV)-based episomal AAV rep-cap plasmid (pCEP-rep/cap). HeLa and 293 cells were stably transfected with plasmids that carry the AAV2 rep/cap genes under transcriptional control of their endogenous promoters (p5, p19 and p40) either on the pCEP-rep/cap or an integrated (pIM45) plasmid. For the ease of monitoring transgene expression in live cells, a rAAV vector expressing gfp (the green fluorescent protein gene, rAAV-gfp/neo) was used. Establishment of stable transfected cell lines with these plasmids proved feasible but their usefulness was limited because of their instability. Within 8-12 weeks after their establishment, stably transfected rep-cap cell lines invariably lost their function. In addition, the rAAV-gfp/neo vector we used was susceptible to mutation in stably transfected HeLa cells. Our observations demonstrate specific problems both at the level of rep/cap gene function and the rAAV genome that can occur with the establishment of rAAV production cell lines. These experiments should aid the further development of efficient rAAV production protocols.

Functional characteristics of S-59 photochemically treated platelet concentrates derived from buffy coats.

BACKGROUND: A photochemical treatment (PCT) process for inactivation of infectious pathogens and leukocytes has been developed and evaluated using single-donor platelet concentrates. This study assessed the application of PCT to platelets prepared from pooled buffy coats. In this study, in vitro functional characteristics of PCT platelets were compared to control platelets prepared from pooled buffy coats using the approved platelet-additive solution T-Sol((R)). Platelets in platelet PAS III additive solution without PCT were evaluated as well. PCT also included the use of a psoralen (S-59) reduction device (SRD). MATERIALS AND METHODS: Four types of platelet concentrates were compared: (1) platelet concentrate in plasma/T-Sol; (2) platelet concentrate in plasma/PAS III; (3) platelet concentrate in plasma/PAS III, PCT, 9 h SRD and (4) platelet concentrate in plasma/PAS III, PCT, 16 h SRD. PCT occurred on the day after whole-blood collection. In vitro assay parameters included: pH, pO(2), pCO(2), HCO(-)(3), platelet count, mean platelet volume, plasma glucose, plasma lactate, total ATP, expression of p-selectin, hypotonic shock response and electron microscopy. RESULTS: The results indicate that PCT is compatible with platelet concentrates prepared from pooled buffy coats for up to 7 days of storage. CONCLUSION: The PCT process resulted in acceptable in vitro platelet functional characteristics and is currently in clinical trials to evaluate the haemostatic efficacy of PCT platelets in thrombocytopenic patients requiring multiple platelet transfusions.

Quality and standardization in blood component preparation with an automated blood processing technique.

The use of automated blood processors in combination with bottom and top blood containers has been found to improve the standardization and quality of blood components. A study was performed to validate a new type of processor (Optipress II) and compare its performance with a first generation processor (Optipress I). Primary separation on the Optipress II was investigated on 570 mL (+/- 10%) of anticoagulated blood in a nonpaired study. In addition, the quality of the products in routine production was compared between the results of the Optipress I and Optipress II. The whole blood units were kept overnight at room temperature (20 +/- 2 degrees C). Separation was performed under conditions to obtain 55 mL buffy coats with a 50% haematocrit (ht). Platelet concentrate preparation was investigated in a paired study and compared to the routine manual method using PAS II additive solution. Parameters studied were volume, red cell, white cell and platelet counts, ht, haemoglobin (hb, total and free). Primary separation was more efficient in the Optipress II because the platelet count was lower in the erythrocyte concentrates (P < 0.0001), platelets were lower in plasma (P < 0.0001) and platelet counts were higher in buffy coats (P < 0.0001). Buffy coat volume showed less variation (Optipress II VC = 4%, Optipress I VC = 7.4%). Secondary separation did not show differences between the Optipress II and manual method but was advantageous because of the automatic termination of the procedure. Further improvement of standardization in blood component preparation is possible with an automated blood processor, leading to improvement of the quality of blood products for patient care.

Beagle model used in a tissue tolerance study of the response of normal and surgically manipulated liver to single high-dose intraoperative radiotherapy.

To investigate the feasibility of delivering a single large dose of intraoperative electron beam radiotherapy (IORT) to the liver of clinically normal and partially hepatectomized beagles, an experimental study was designed. The purpose of the study was to obtain dose guidelines for the delivery of IORT to the liver of human patients with colorectal cancer metastases to the liver. After partial resection of the liver, IORT in doses up to 30 Gy was applied to the resection plane as well as to a nonsurgically manipulated part of the liver of 25 beagles. The temporal sequence of histologic changes of these irradiated parts of the liver tissue was investigated. There were no postoperative complications and no morbidity or mortality associated with a minimal follow-up of 3 years. Necropsy performed 3 months after IORT revealed only mild histopathologic changes. One year after IORT, more distinct histopathologic changes consisting of capsular thickening, diffuse parenchymal fibrosis, and subcapsular hepatocellular atrophy were found. Three years after IORT, the parenchymal architecture seemed to be restored, although loss of liver tissue was definitive at the irradiation site; liver function remained intact. These results indicate that IORT to part of the liver in the canine model can be safely applied and that, although doses up to 30 Gy can result in severe local tissue damage, wound healing and liver function are not disturbed.

Expression of the jun family of genes in human ovarian cancer and normal ovarian surface epithelium.

The jun genes (c-jun, jun-B and jun-D) play a role in critical cell functions such as proliferation, differentiation and apoptosis. We documented jun expression at the mRNA and protein level in human ovarian cancer tissues (n=28), surface epithelial cells of normal ovaries (n=14) and ovarian cancer cell lines (n=6). Almost all of ovarian tumors as well as normal ovaries concomitantly express c-jun, jun-B, and jun-D mRNA. Immunohistochemistry was less sensitive and revealed nuclear c-Jun and Jun-B proteins in the malignant epithelial cells of respectively 38% and 11% of ovarian tumors and in the surface epithelium of a normal premenopausal ovary. In cultured ovarian cancer cells, c-jun and jun-B expression is inducible by serum and TPA and is therefore not constitutive. The c-jun and jun-B proteins therefore play a role both in differentiation of the normal ovarian surface epithelium, as well as in the proliferation of epithelial ovarian cancer cells. High jun-B expression relates to a more malignant phenotype both in vitro and in vivo. The jun-D gene is suppressed in ovarian cancer cells as compared to normal ovarian surface epithelial cells in situ and in vitro. Downregulation of jun-D might therefore be part of the malignant ovarian epithelial cell phenotype.

Tissue tolerance of normal and surgically manipulated canine liver to intraoperative radiation therapy (IORT).

PURPOSE: The purpose of the study is to obtain dose guidelines for the delivery of intraoperative radiotherapy to the liver of patients with colorectal liver metastases. Following partial resection of the liver, a single high dose of 10, 20, 25, and 30 Gy intraoperative radiotherapy was applied to both the resection plane as well as a nonsurgically manipulated part of the liver of 25 beagles. The temporal sequence of histological and ultrastructural changes of these irradiated parts of the liver tissue was investigated. METHODS AND MATERIALS: The feasibility of delivering single large dose of intraoperative electron beam radiotherapy to the normal and partially hepatectomized liver was experimentally investigated in a canine study. RESULTS: There were no postoperative complications, no morbidity or mortality with a minimal follow-up of 1 year. Autopsy performed 3 months following irradiation showed only mild histopathological changes. One year following intraoperative radiotherapy more distinct histopathological changes consisting of capsular thickening, diffuse parenchymal fibrosis and subcapsular hepatocellular atrophy were found. The liver function remained intact. CONCLUSION: This study demonstrated that intraoperative radiotherapy to part of the liver in the canine model can be safely applied and doses up to 30 Gy are well tolerated.

Accumulation and localization of gallium-67 in various types of primary lung carcinoma.

The uptake and location of Ga-67 were investigated in 15 primary pulmonary carcinomas. The accumulation in the tumor was determined by scintigraphy of the patient, grain counts over fields of tumor cells in autoradiographs of tumor-tissue samples, and gamma counts in specimens of the tumor. Good correlation was found between the results obtained with these three methods. The relationship between accumulation of Ga-67 in the tumor and the histologic type of tumor was also studied. Undifferentiated carcinomas, and tumor cells in squamous-cell carcinomas showed significantly more Ga-67 than tumor cells in adenocarcinomas. No correlation was found between the presence of inflammatory infiltrates in or around the tumor and the grade of the scintigraphic images. In the autoradiograms, lymphocytes, plasma cells, granulocytes, and macrophages showed less radioactivity than the tumor cells--or none at all. Collagen fibers appeared to have bound some Ga-67, but necrotic areas showed no uptake.

Total skin electron irradiation in mycosis fungoides.

Total skin irradiation with fast electrons (4 MeV, 3,500 rad) was applied in 10 cases of histologically proven mycosis fungoides without any signs of internal dissemination. The technique is described. Complete disappearance of both lesions and symptoms occurred in all patients after treatment. However, half of the patients relapsed after the initial irradiation and were subsequently treated with a booster dose of 1,000 rad 4 MeV electrons or with topical nitrogen mustard. Irradiation with fast electrons seems to be of great importance in the treatment of mycosis fungoides.