Improving molecular testing and personalized medicine in non-small-cell lung cancer in Ontario.

BACKGROUND: Although molecular testing has become standard in managing advanced nonsquamous non-small-cell lung cancer (nsclc), most patients undergo minimally invasive procedures, and the diagnostic tumour specimens available for testing are usually limited. A knowledge translation initiative to educate diagnostic specialists about sampling techniques and laboratory processes was undertaken to improve the uptake and application of molecular testing in advanced lung cancer. METHODS: A multidisciplinary panel of physician experts including pathologists, respirologists, interventional thoracic radiologists, thoracic surgeons, medical oncologists, and radiation oncologists developed a specialty-specific education program, adapting international clinical guidelines to the local Ontario context. Expert recommendations from the program are reported here. RESULTS: Panel experts agreed that specialists procuring samples for lung cancer diagnosis should choose biopsy techniques that maximize tumour cellularity, and that conservation strategies to maximize tissue for molecular testing should be used in tissue processing. The timeliness of molecular reporting can be improved by pathologist-initiated reflex testing upon confirmation of nonsquamous nsclc and by prompt transportation of specimens to designated molecular diagnostic centres. To coordinate timely molecular testing and optimal treatment, collaboration and communication between all clinicians involved in diagnosing patients with advanced lung cancer are mandatory. CONCLUSIONS: Knowledge transfer to diagnostic lung cancer specialists could potentially improve molecular testing and treatment for advanced lung cancer patients.

Prognostic value of pretreatment circulating neutrophils, monocytes, and lymphocytes on outcomes in lung stereotactic body radiotherapy.

PURPOSE: In the present study, we determined the association of pretreatment circulating neutrophils, monocytes, and lymphocytes with clinical outcomes after lung stereotactic body radiotherapy (sbrt). METHODS: All patients with primary lung cancer and with a complete blood count within 3 months of lung sbrt from 2005 to 2012 were included. Overall survival (os) was calculated using the Kaplan-Meier method. Factors associated with os were investigated using univariable and multivariable Cox proportional hazards regression. Fine-Gray competing risk regression was performed to test the association of the neutrophil:lymphocyte (nlr) and monocyte:lymphocyte (mlr) ratios with two types of failure: disease-related failure and death, and death unrelated to disease. RESULTS: Of the 299 sbrt patients identified, 122 were eligible for analysis. The median and range of the nlr and mlr were 3.0 (0.3-22.0) and 0.4 (0.1-1.9) respectively. On multivariable analysis, sex (p = 0.02), T stage (p = 0.04), and nlr (p < 0.01) were associated with os. On multivariable analysis, T stage (p < 0.01) and mlr (p < 0.01) were associated with disease-related failure; mlr (p = 0.03), nlr (p < 0.01), and sbrt dose of 48 Gy in 4 fractions (p = 0.03) and 54 Gy or 60 Gy in 3 fractions (p = 0.02) were associated with disease-unrelated death. Median survival was 4.3 years in the nlr≤3 group (95% confidence interval: 3.5 to not reached) and 2.5 years in the nlr>3 group (95% confidence interval: 1.7 to 4.8; p < 0.01). CONCLUSIONS: In lung sbrt patients, nlr and mlr are independently associated with os and disease-unrelated death. If validated, nlr and mlr could help to identify patients who would benefit most from sbrt.

Metabolic syndrome-related hepatocellular carcinoma treated by volumetric modulated arc therapy.

Hepatocellular carcinoma (hcc) is a leading cause of cancer mortality, and its incidence is increasing in developed countries. Risk factors include cirrhosis from viral hepatitis or alcohol abuse. Metabolic syndrome is a newly recognized, but important, risk factor that is likely contributing to the increased incidence of hcc. Surgery is the therapy of choice for hcc, but local therapies are often contraindicated, usually because of advanced disease or comorbid conditions such as cardiac disease (which is associated with metabolic syndrome). Current radiation therapy techniques such as stereotactic body radiotherapy allow for treatment plans that highly conform to the target and provide excellent sparing of normal structures. Radiation therapy is emerging as a viable option in patients not eligible for surgery or other locoregional therapies. Here, we report a case of a large hcc presenting in a patient with metabolic syndrome without significant alcohol history or biochemical liver dysfunction. The patient was not a candidate for locoregional therapies because of cardiac and renal comorbidities typical of patients experiencing the long-term sequelae of metabolic syndrome. Treatment using an arc-based volumetric-modulated arc therapy technique allowed for the highest dose of radiation to be delivered to the tumour while the peripheral radiation dose was minimized. A complete local response was confirmed by computed tomography imaging 21 months after treatment completion.

Perspectives on treatment advances for stage III locally advanced unresectable non-small-cell lung cancer.

For more than a decade, there has been no improvement in outcomes for patients with unresectable locally advanced (la) non-small-cell lung cancer (nsclc). The standard treatment in that setting is definitive concurrent chemotherapy and radiation (ccrt). Although the intent of treatment is curative, most patients rapidly progress, and their prognosis is poor, with a 5-year overall survival (os) rate in the 15%-25% range. Those patients therefore represent a critical unmet need, warranting expedited approval of, and access to, new treatments that can improve outcomes. The pacific trial, which evaluated durvalumab consolidation therapy after ccrt in unresectable la nsclc, demonstrated a statistically significant and clinically meaningful improvement in progression-free survival (pfs) and a significant improvement in os. Durvalumab thus fills a critical unmet need in the setting of unresectable la nsclc and provides a new option for patients treated with curative intent. Here, we review the treatment of unresectable la nsclc, with a focus on the effect of the clinical data for durvalumab.

Differential chemosensitivity of breast cancer cells to ganciclovir treatment following adenovirus-mediated herpes simplex virus thymidine kinase gene transfer.

The development of resistance to radiation and chemotherapeutic agents that cause DNA damage is a major problem for the treatment of breast and other cancers. The p53 tumor suppressor gene plays a direct role in the signaling of cell cycle arrest and apoptosis in response to DNA damage, and p53 gene mutations have been correlated with increased resistance to DNA-damaging agents. Herpes simplex virus thymidine kinase (HSV-tk) gene transfer followed by ganciclovir (GCV) treatment is a novel tumor ablation strategy that has shown good success in a variety of experimental tumor models. However, GCV cytotoxicity is believed to be mediated by DNA damage-induced apoptosis, and the relationship between p53 gene status, p53-mediated apoptosis, and the sensitivity of human tumors to HSV-tk/GCV treatment has not been firmly established. To address this issue, we compared the therapeutic efficacy of adenovirus-mediated HSV-tk gene transfer and GCV treatment in two human breast cancer cell lines: MCF-7 cells, which express wild-type p53, and MDA-MB-468 cells, which express high levels of a mutant p53 (273 Arg-His). Treating MCF-7 cells with AdHSV-tk/GCV led to the predicted increase in endogenous p53 and p21WAF1/CIP1 protein levels, and apoptosis was observed in a significant proportion of the target cell population. However, treating MDA-MB-468 cells under the same conditions resulted in a much stronger apoptotic response in the absence of induction in p21WAF1/CIP1 protein levels. This latter result suggested that HSV-tk/GCV treatment can activate a strong p53-independent apoptotic response in tumor cells that lack functional p53. To confirm this observation, four additional human breast cancer cell lines expressing mutant p53 were examined. Although a significant degree of variability in GCV chemosensitivity was observed in these cell lines, all displayed a greater reduction in cell viability than MCF-7 or normal mammary cells treated under the same conditions. These results suggest that endogenous p53 status does not correlate with chemosensitivity to HSV-tk/GCV treatment. Furthermore, evidence for a p53-independent apoptotic response serves to extend the potential of this therapeutic strategy to tumors that express mutant p53 and that may have developed resistance to conventional genotoxic agents.

Heat-directed gene targeting of adenoviral vectors to tumor cells.

Targeting therapeutic gene expression to tumor cells represents a major challenge for cancer gene therapy. The strong transcriptional response exhibited by heat shock genes, along with the beneficial therapeutic effects of hyperthermia have led us to develop a heat-directed gene-targeting strategy for cancer treatment. Heat shock gene expression is mediated in large part by the interaction of heat shock factor 1 with specific binding sites (heat shock elements; HSE) found in the promoters of heat-inducible genes. Here we present a quantitative analysis of heat-inducible gene expression mediated by the wild-type hsp70b gene promoter, as well as a modified hsp70b promoter containing additional HSE sequences. Beta-galactosidase (beta-gal) expression was induced between 50- and 800-fold in a panel of human breast cancer cell lines infected with an adenoviral vector containing the wild-type hsp70b promoter (Ad.70b.betag) following treatment at 43 degrees C for 30 minutes. Infection with an adenoviral vector containing the modified hsp70b promoter (Ad.HSE.70b.betag) resulted in a 200- to 950-fold increase in beta-gal expression under the same conditions, and also provided a 1-2 degrees C decrease in the threshold of activation. Significant increases in the heat responsiveness of the Ad.HSE.70b.betag construct were observed in five of six tumor cell lines tested, as well as under thermotolerant conditions. Finally, we demonstrate that localized heating of a HeLa cell xenograft can effectively target beta-gal gene expression following intratumoral injection of Ad.70b.betag. Adenoviral vectors incorporating heat-inducible therapeutic genes may provide useful adjuncts for clinical hyperthermia.

Induction gemcitabine plus concurrent gemcitabine and radiotherapy for locally advanced unresectable or resected pancreatic cancer.

AIMS: To determine the efficacy of induction gemcitabine followed by biweekly gemcitabine concurrent with radiotherapy for locally advanced pancreatic cancer. MATERIALS AND METHODS: Between March 2001 and August 2009, 90 patients with unresectable (78) or resected (12) pancreatic cancer were treated with a standard treatment policy of induction gemcitabine (seven doses of weekly gemcitabine at 1000 mg/m(2)) followed by concurrent radiotherapy (52.5 Gy) and biweekly gemcitabine (40 mg/m(2)). RESULTS: After induction gemcitabine, 17.8% of patients did not proceed to chemoradiotherapy, due to either disease progression, performance status deterioration or gemcitabine toxicity. Of the patients who received chemoradiotherapy, 68.9% completed the course of 52.5 Gy, whereas 79.7% received more than 45 Gy. Chemoradiotherapy was stopped early due to treatment toxicity in 22.9% of patients. On intention to treat analysis, the median overall survival was 12.7 months in the locally advanced group and 18.2 months in the resected group. On multivariate analysis for the unresectable patients, a larger gross tumour volume was a significant poor prognostic factor for overall survival and local progression-free survival. CONCLUSION: This large series confirms, in a standard practice setting, similar efficacy and tolerability of treatment as previously reported in our phase I-II study. The benefit to patients with a gross tumour volume >48 cm(3) may be limited.

Outcomes of accelerated hypofractionated radiotherapy in stage i non-small-cell lung cancer.

PURPOSE: Outcomes after treatment with accelerated hypofractionated radiotherapy in stage i medically inoperable non-small-cell lung cancer (nsclc) patients were determined. METHODS: Our single-institution retrospective review looked at medically inoperable patients with T1-2N0M0 nsclc treated with accelerated hypofractionated curative-intent radiotherapy between 1999 and 2009. Patients were staged mainly by computed tomography imaging of chest and abdomen, bone scan, and computed tomography/magnetic resonance imaging of brain. Positron-emission tomography (pet) staging was performed in 6 patients. Medical charts were reviewed to determine demographics, radiotherapy details, sites of failure, toxicity (as defined by the Common Terminology Criteria for Adverse Events, version 3.0) and vital status. The cumulative incidence of local and distant failure was calculated. Overall (os) and cause-specific (css) survival were estimated by the Kaplan-Meier method. RESULT: In the 60 patients treated during the study period, the dose regimens were 50 Gy in 20 fractions (n = 6), 55 Gy in 20 fractions (n = 8), 60 Gy in 20 fractions (n = 42), and 60 Gy in 25 fractions (n = 4). All patients were treated once daily. The median follow-up was 27 months (range: 4-94 months). The os rates at 2 and 5 years were 61% [95% confidence interval (ci): 50% to 75%] and 19% (95% ci: 10% to 34%) respectively. The css rates at 2 and 5 years were 79% (95% ci: 68% to 91%) and 39% (95% ci: 24% to 63%) respectively. The cumulative incidence of local failure was 20% at 5 years. The cumulative incidence of distant failure was 28% at 5 years. No patients experienced grade 3 or greater pneumonitis or esophagitis. CONCLUSIONS: Accelerated hypofractionated regimens are well tolerated and provide good local control in medically inoperable patients with stage i nsclc. Such regimens may be a reasonable treatment alternative when stereotactic body radiation therapy is not feasible.

Early metabolic response evaluation after stereotactic radiotherapy for lung cancer: pilot experience with 18F-fluorodeoxyglucose positron emission tomography-computed tomography.

The early response of lung tumours to stereotactic radiotherapy was prospectively evaluated with 18F-fluorodeoxyglucose positron emission tomography-computed tomography. Three months after treatment, the maximum standardised uptake value and the tumour diameter fell by 64 and 30%, respectively. This imaging strategy therefore remains under ongoing evaluation with the aim of identifying predictive and prognostic factors.

[Folk Medicine has many universal remedies - the most important is "Aquavit"]. / Et højst simpelt og pålideligt universal-laegemiddel.

In 1875 A.P. Møller, a bookseller on "Gammelmønt" in Copenhagen published a little book on "aquavit" as a universal medicament written by an old shepherd from Schlesien. The idea behind the book is discussed and several additions to aquavit are mentioned. The adding of salt is especially recommended for many ailments. The Danes have generally prefered to add other substances, which is shown by comparing the shepherds list with a danish investigation.

[Anatomical investigations on the lower leg and foot in cases of hypoplasia and aplasia of the tibia (author's transl)]. / Anatomische Untersuchungen an Unterschenkel und Fuss bei Hypoplasie und Aplasie der Tibia.

Deformities associated with absence or defect of the tibia were investigated on 3 lower legs and feet. In case of bilateral hypoplasia of the tibia, the deformities are nearly identical regarding the muscles and the skeletal system. The main vessels and nerves are developed normally. The extent of malformations seems to be greater in case of aplasia of the tibia. The question, wether some abnormalities typical for a defect of the tibia exist, is discussed. For this our own observations are compared with similar cases described in the literature.

[Migration of Kirschner wire following osteosyntheses of the hip (author's transl)]. / Kirschnerdrahtwanderung nach Osteosynthesen an der Hüfte.

The article reports on two cases of migrations of Kirschner wire following osteosyntheses at the hip joint. In one case of epiphyseodesis, the steel wire for skeletal transfixion was bent and eventually broken. Seven months later, a 6 cm piece of wire had to be removed from the omentum majus of the 14-year-old male patient concerned. In a child aged 4 years, coxa vara congenita was established intertrochanterally and osteotomy fixed by means of a shackle screw after Pohl. For greater safety, a Kirschner wire was additionally introduced. The wire migrated as far as the anterior abdominal wall. The article discusses guidelines which should be observed when employing Kirschner wires.

[Histological findings of the femoral quadriceps muscle in varus and valgus gonarthrosis]. / Histologische Befunde am M. quadriceps femoris bei Varus- und Valgusgonarthrose.

A pronounced valgization or varization of the lower extremity leads to changes in skeletal muscle which are histologically and morphometrically demonstrable in biopsies of Mm. vastus lateralis et medialis of patients with chronically anomalous stressing of the knee joint. During chronic over-stressing, increased thickness in type I and type II muscle fibers can be observed in both Mm. vastus medialis and lateralis, whereas type II fibers demonstrate a greater variability and there is a greater occurrence in type I fibers. Our investigations show that a chronic anomalous stressing influences both the size and energy metabolism of a skeletal muscle cell in a manner which can be interpreted as a functional adaptation.

[Niels Finsen's treatment of tuberculosis of the skin]. / Niels Finsens behandling af hudtuberkulose.

By his contemporaries Niels Ryberg Finsen (1860-1904) has been described as a highly dynamic personality and characterised as a sympathetic, kind, confidence-inspiring, conscientious, reliable, intellectual, mysterious and genious doctor, who took care of his patients' illness as well as their social and economic conditions. But he was known too as sharp, critical and able to defend his scientific viewpoints with a deep commitment. Niels Finsen introduced the universal light bath, but his great achievement was the local light treatment of lupus vulgaris for which he got the Nobel Prize in 1903. In the article the history of lupus vulgaris, its treatment and its social consequenses for the patients as individuals are described as an introduction to an analysis of Niels Finsen's scientific methods, his treatment and results and the reactions from the contemporary medical authorities, the public represented by the press and the patients themselves. Finally the discussions after Niels Finsen's death are researched.

Astrocyte reactivity in neonatal mice: apparent dependence on the presence of reactive microglia/macrophages.

In neonatal mice, an acute injury produced by a stab wound to the cortex results in minimal astrocyte reactivity, as has been observed by others. However, if the source of the stab wound, a piece of nitrocellulose (NC) membrane, were now implanted in the cortex for a period of time (chronic NC implant injury), then extensive astroglial reactivity in the neonatal brain ensues. The astrogliosis is manifested by increased mRNA, protein content, and immunoreactivity for GFAP, and by ultrastructural changes. Given the previous reports that inflammatory cytokines are possible mediators of astrocyte reactivity (e.g., Balasingam et al: J Neurosci 14:846, 1994), we examined the brain parenchyma of neonatal mice following an NC stab or implant injury, with minimal or extensive astrogliosis, respectively, for a possible differential representation of inflammatory cells. A significant correlation (r = 0.87, P < 0.05) was observed between the occurrence of astrogliosis and the presence of reactive microglia/macrophages; no other inflammatory cell type was detected in the brain parenchyma of neonatal mice following NC implant injury. We suggest that reactive microglia/macrophages are required for the evolution of cells into reactive astrocytes following insults to the neonatal brain.

Peroxisome biogenesis in yeast.

Eukaryotic cells have evolved a complex set of intracellular organelles, each of which possesses a specific complement of enzymes and performs unique metabolic functions. This compartmentalization of cellular functions provides a level of metabolic control not available to prokaryotes. However, it presents the eukaryotic cell with the problem of targeting proteins to their specific location(s). Proteins must be efficiently transported from their site of synthesis in the cytosol to their specific organelle(s). Such a process may require translocation across one or more hydrophobic membrane barriers and/or asymmetric integration into specific membranes. Proteins carry cis-acting amino acid sequences that serve to act as recognition motifs for protein sorting and for the cellular translocation machinery. Sequences that target proteins to the endoplasmic reticulum/secretory pathway, mitochondria, and chloroplasts are often present as cleavable amino-terminal extensions. In contrast, most peroxisomal proteins are synthesized at their mature size and are translocated to the organelle without any post-translational modification. This review will summarize what is known about how yeast solve the problem of specifically importing proteins into peroxisomes and will suggest future directions for investigations into peroxisome biogenesis in yeast.

Placental transforming growth factor-beta is a downstream mediator of the growth arrest and apoptotic response of tumor cells to DNA damage and p53 overexpression.

The p53 tumor suppressor gene and members of the transforming growth factor-beta (TGF-beta) superfamily play central roles in signaling cell cycle arrest and apoptosis (programmed cell death) in normal development and differentiation, as well as in carcinogenesis. Here we describe a distantly related member of the TGF-beta superfamily, designated placental TGF-beta (PTGF-beta), that is up-regulated in response to both p53-dependent and -independent apoptotic signaling events arising from DNA damage in human breast cancer cells. PTGF-beta is normally expressed in placenta and at lower levels in kidney, lung, pancreas, and muscle but could not be detected in any tumor cell line studied. The PTGF-beta promoter is activated by p53 and contains two p53 binding site motifs. Functional studies demonstrated that one of these p53 binding sites is essential for p53-mediated PTGF-beta promoter induction and specifically binds recombinant p53 in gel mobility shift assays. PTGF-beta overexpression from a recombinant adenoviral vector (AdPTGF-beta) led to an 80% reduction in MDA-MB-468 breast cancer cell viability and a 50-60% reduction in other human breast cancer cell lines studied, including MCF-7 cells, which are resistant to growth inhibition by recombinant wild-type p53. Like p53, PTGF-beta overexpression was seen to induce both G(1) cell cycle arrest and apoptosis in breast tumor cells. These results provide the first evidence for a direct functional link between p53 and the TGF-beta superfamily and implicate PTGF-beta as an important intercellular mediator of p53 function and the cytostatic effects of radiation and chemotherapeutic cancer agents.

PAY4, a gene required for peroxisome assembly in the yeast Yarrowia lipolytica, encodes a novel member of a family of putative ATPases.

PAY genes are required for peroxisome assembly in the yeast Yarrowia lipolytica. Here we characterize one mutant, pay4, and describe the cloning and sequencing of the PAY4 gene. The pay4 mutant shows no identifiable peroxisomes by biochemical and morphological criteria. The complementing PAY4 gene encodes a polypeptide, Pay4p, 1025 amino acids in length and having a predicted molecular mass of 112,258 Da. The predicted Pay4p sequence contains two putative ATP-binding domains and shows structural relationships to other potential ATP-binding proteins involved in biological processes as diverse as peroxisome biogenesis, vesicle-mediated protein transport, cell cycle control, and transcriptional regulation. These proteins all share a highly conserved stretch of approximately 175 amino acids that contains a consensus sequence for ATP binding. Pay4p shows sequence conservation with Pas1p and Pas5p, putative ATPases required for peroxisomal assembly in the yeasts Saccharomyces cerevisiae and Pichia pastoris, respectively. Pay4p, Pas1p, and Pas5p are presumably related members of a family of putative ATPases involved in peroxisome biogenesis. Pay4p is synthesized in low amounts in Y. lipolytica cells grown in glucose, and there is a rapid and pronounced increase in the levels of Pay4p upon transfer of the cells to a medium containing oleic acid as the sole carbon source.