Implementing multiplexed genotyping of non-small-cell lung cancers into routine clinical practice.

BACKGROUND: Personalizing non-small-cell lung cancer (NSCLC) therapy toward oncogene addicted pathway inhibition is effective. Hence, the ability to determine a more comprehensive genotype for each case is becoming essential to optimal cancer care. METHODS: We developed a multiplexed PCR-based assay (SNaPshot) to simultaneously identify >50 mutations in several key NSCLC genes. SNaPshot and FISH for ALK translocations were integrated into routine practice as Clinical Laboratory Improvement Amendments-certified tests. Here, we present analyses of the first 589 patients referred for genotyping. RESULTS: Pathologic prescreening identified 552 (95%) tumors with sufficient tissue for SNaPshot; 51% had ≥1 mutation identified, most commonly in KRAS (24%), EGFR (13%), PIK3CA (4%) and translocations involving ALK (5%). Unanticipated mutations were observed at lower frequencies in IDH and ß-catenin. We observed several associations between genotypes and clinical characteristics, including increased PIK3CA mutations in squamous cell cancers. Genotyping distinguished multiple primary cancers from metastatic disease and steered 78 (22%) of the 353 patients with advanced disease toward a genotype-directed targeted therapy. CONCLUSIONS: Broad genotyping can be efficiently incorporated into an NSCLC clinic and has great utility in influencing treatment decisions and directing patients toward relevant clinical trials. As more targeted therapies are developed, such multiplexed molecular testing will become a standard part of practice.

Gene expression in histologically normal epithelium from breast cancer patients and from cancer-free prophylactic mastectomy patients shares a similar profile.

INTRODUCTION: We hypothesised that gene expression in histologically normal (HN) epithelium (NlEpi) would differ between breast cancer patients and usual-risk controls undergoing reduction mammoplasty (RM), and that gene expression in NlEpi from cancer-free prophylactic mastectomy (PM) samples from high-risk women would resemble HN gene expression. METHODS: We analysed gene expression in 73 NlEpi samples microdissected from frozen tissue. In 42 samples, we used microarrays to compare gene expression between 18 RM patients and 18 age-matched HN (9 oestrogen receptor (ER)+, 9 ER-) and 6 PM patients. Data were analysed using a Bayesian approach (BADGE), and validated with quantitative real-time PCR (qPCR) in 31 independent NlEpi samples from 8 RM, 17 HN, and 6 PM patients. RESULTS: A total of 98 probe sets (86 genes) were differentially expressed between RM and HN samples. Performing hierarchical analysis with these 98 probe sets, PM and HN samples clustered together, away from RM samples. qPCR validation of independent samples was high (84%) and uniform in RM compared with HN patients, and lower (58%), but more heterogeneous, in RM compared with PM patients. The 86 genes were implicated in many processes including transcription and the MAPK pathway. CONCLUSION: Gene expression differs between the NlEpi of breast cancer cases and controls. The profile of cancer cases can be discerned in high-risk NlEpi from cancer-free breasts. This suggests that the profile is not an effect of the tumour, but may mark increased risk and reveal the earliest genomic changes of breast cancer.

Distinct mechanisms of nonhomologous end joining in the repair of site-directed chromosomal breaks with noncomplementary and complementary ends.

DNA double-strand breaks (DSBs) are considered the most important type of DNA damage inflicted by ionizing radiation. The molecular mechanisms of DSB repair by nonhomologous end joining (NHEJ) have not been well studied in live mammalian cells, due in part to the lack of suitable chromosomal repair assays. We previously introduced a novel plasmid-based assay to monitor NHEJ of site-directed chromosomal I-SceI breaks. In the current study, we expanded the analysis of chromosomal NHEJ products in murine fibroblasts to focus on the error-prone rejoining of DSBs with noncomplementary ends, which may serve as a model for radiation damage repair. We found that noncomplementary ends were efficiently repaired using microhomologies of 1-2 nucleotides (nt) present in the single-stranded overhangs, thereby keeping repair-associated end degradation to a minimum (2-3 nt). Microhomology-mediated end joining was disrupted by Wortmannin, a known inhibitor of DNA-PKcs. However, Wortmannin did not significantly impair the proficiency of end joining. In contrast to noncomplementary ends, the rejoining of cohesive ends showed only a minor dependence on microhomologies but produced fivefold larger deletions than the repair of noncomplementary ends. Together, these data suggest the presence of several distinct NHEJ mechanisms in live cells, which are characterized by the degree of sequence deletion and microhomology use. Our NHEJ assay should prove a useful system to further elucidate the genetic determinants and molecular mechanisms of site-directed DSBs in living cells.

Monitoring of biological odour filtration in closed environments with olfactometry and an electronic nose.

Air treatment with a compact biological membrane filter, and air quality monitoring with an electronic nose were tested in the laboratory on air from a cage containing six mice. Additional analyses of air to and from the filter were performed using olfactometry and ammonia and hydrogen sulphide gas detection tubes. The biological air filter is a module containing biofilm-coated membrane fibres that separate a closed liquid loop from a gas phase. Odour compounds and oxygen diffuse through the membranes from the gas phase to the biofilm, where they are degraded to carbon dioxide and water. The prototype "ENQBE" electronic nose is based on an array of eight thickness shear mode resonators (TSMR), also known in the literature as quartz microbalance sensors. The chemical sensitivity is given by molecular films of metalloporphyrins and similar compounds. Chemical interaction of compounds in the air with the vibrating sensors induces a frequency change of the vibration that can be measured as a signal. The air from the mouse cage had a strong odour (3490 OUE/m3). The biological membrane filter performed well, achieving over 80% odour and ammonia reduction. The electronic nose signal could be correlated with the inlet and outlet air-quality of the biological filter, making it a promising method for monitoring air quality in closed environments.

Odour abatement in the integrated reactor concept for simultaneous treatment of liquid and solid pig manure fractions.

New technologies are needed for manure treatment that can capture nutrients, reduce emissions of ammonia and nuisance odours, and kill harmful pathogens. A reactor concept was developed for simultaneous treatment of separately collected liquid and solid fractions of pig manure. The liquid fraction is concentrated by evaporating the water using energy from the composting system and at the same time scrubbing ammonia from the composting off-gas by acidifying the urine to pH 4 with nitric acid. This results in two marketable products, a concentrated liquid nitrogen fertiliser (NH4NO3) without phosphorus, and a stabilised, solid organic fertiliser, which is free of pathogens and weeds. By connecting the two reactor systems, emissions of ammonia and odours are abated as ammonia emitted from the composting is trapped in the liquid fraction and odorous compounds emitted from the liquid are degraded in the composting reactor. The concept was physically simulated by coupling a 80-L compost reactor to a 10-L bubble column. Operation of the bench-scale system showed that the concept is very promising. All nutrients were captured, and emissions of ammonia and odours were almost completely abated.

Repair of radiation damage to DNA.

DNA double-strand breaks constitute the most dangerous type of DNA damage induced by ionising radiation (IR). Accordingly, the resistance of cells to IR is modulated by three intimately related cellular processes: DNA repair, recombination, and replication. Significant discoveries in this field of research have been made over the last few years. A picture seems to be emerging in which perturbations of recombination in cancer cells are a more widespread cause of genomic instability than previously appreciated. Conversely, such cells may also be more sensitive to certain chemotherapeutic drugs and to IR. Thus, the alterations in recombination that promote carcinogenesis by causing genomic instability may also be the weakness of the tumours that arise in this setting, a concept which could hold great promise for the advancement of cancer treatment in the not too distant future.

Homologous recombination in extrachromosomal plasmid substrates is not suppressed by p53.

We and others reported previously that the tumor suppressor p53 down-regulates spontaneous homologous recombination in chromosomally integrating plasmid substrates, but how p53 affects homology-dependent repair of DNA double-strand breaks has not been established. Furthermore, it has been hypothesized that p53 may suppress homologous recombination by direct interaction with recombination intermediates, but it is not known whether p53 directly acts on extrachromosomal plasmid substrates. In the present study, we asked whether p53 can suppress extrachromosomal spontaneous and double-strand break-induced homologous recombination. A plasmid shuttle assay was employed utilizing episomally replicating substrates, which carried mutated tandem repeats of a CAT reporter gene. Spontaneous homologous recombination and homology-dependent repair of double-strand breaks induced by the I-SceI nuclease led to reconstitution of the reporter. Extrachromosomal homologous recombination was found to proceed independently of the p53 status of isogenic mouse fibroblast lines, contrasting the p53-mediated suppression of chromosomal recombination. The lack of p53 effect applied not only to the dominating single-strand annealing pathway, which is Rad51-independent, but also to Rad51-dependent gene conversion events. Comparison of homologous and non-homologous recombination frequencies revealed similar contributions to the repair of I-SceI-induced breaks irrespective of p53 status. Our results are consistent with a model in which the regulation of homologous recombination by p53 is restricted to the highly ordered chromosomal chromatin structure. These data may serve as a cautionary note for future investigations using solely extrachromosomal model systems to address DNA repair in intact cells.

Deficiency of human BRCA2 leads to impaired homologous recombination but maintains normal nonhomologous end joining.

Carriers of BRCA2 germline mutations are at high risk to develop early-onset breast cancer. The underlying mechanisms of how BRCA2 inactivation predisposes to malignant transformation have not been established. Here, we provide direct functional evidence that human BRCA2 promotes homologous recombination (HR), which comprises one major pathway of DNA double-strand break repair. We found that up-regulated HR after transfection of wild-type (wt) BRCA2 into a human tumor line with mutant BRCA2 was linked to increased radioresistance. In addition, BRCA2-mediated enhancement of HR depended on the interaction with Rad51. In contrast to the tumor suppressor BRCA1, which is involved in multiple DNA repair pathways, BRCA2 status had no impact on the other principal double-strand break repair pathway, nonhomologous end joining. Thus, there exists a specific regulation of HR by BRCA2, which may function to maintain genomic integrity and suppress tumor development in proliferating cells.

Loss of wild-type p53 function is responsible for upregulated homologous recombination in immortal rodent fibroblasts.

PURPOSE: A correlation between mutations in the tumour suppressor gene p53 and high rates of homologous recombination were previously found in immortal rodent fibroblasts. In the current study, direct evidence was sought that loss of p53 function is mainly responsible for upregulated levels of homologous recombination. MATERIALS AND METHODS: Homologous recombination was assessed in vitro using DNA plasmid substrates that stably integrated into the genome of mouse and rat embryonic fibroblasts. RESULTS: Primary fibroblasts with wild-type p53 displayed a recombination rate of about 1 x 10(-4). This number increased by 33- to 93-fold after spontaneous cellular immortalization, accompanied by loss of p53 function. To exclude potential bias from other gene mutations, wild-type p53 was experimentally disrupted in primary fibroblasts leading to an increase in recombination by one order of magnitude. Conversely, re-introduction of wild-type p53 into p53-null immortal cells reconstituted suppressed recombination rates. Finally, early-passage fibroblast cultures from p53-knock-out mice showed elevated recombination rates, which did not increase further following immortalization. CONCLUSIONS: Loss of wild-type p53 is the major genetic determinant of increased homologous recombination frequencies in immortal rodent fibroblasts. Cellular p53 status will be an important factor to consider when performing functional analysis of the increasing number of mammalian proteins that are found to be involved in homologous recombination.

Dissociation of p53-mediated suppression of homologous recombination from G1/S cell cycle checkpoint control.

The tumor suppressor p53 is considered as the guardian of the genome which is activated following genotoxic stress. In many cell types, p53 mediates G1 cell cycle arrest as the predominant cellular response. Inactivation of wild-type p53 leads to loss of G1/S checkpoint control and to genomic instability, including increased spontaneous homologous recombination (HR). To determine whether regulation of the G1/S checkpoint is required for suppression of HR, we assessed recombination events using a plasmid substrate that stably integrated into the genome of p53-null mouse fibroblasts. Exogenous expression of a temperature-sensitive p53 protein (Ala135 to Val), which had lost trans-activation function and could not regulate G1/S transition when in mutant conformation, reduced HR rates to the same extent as wild-type p53. Furthermore, a p53 construct with an alternatively-spliced carboxy terminus also retained this ability in the absence of both activities, G1/S control and non-sequence specific DNA binding as mediated by the carboxy terminus. Our data dissociate regulation of HR by p53 from its role as a cell cycle checkpoint protein. The results support a model which extends p53's role as a guardian of the genome to include transactivation-independent regulatory functions in DNA repair, replication and recombination.

p53 directly enhances rejoining of DNA double-strand breaks with cohesive ends in gamma-irradiated mouse fibroblasts.

The p53 gene regulates the cell cycle response to DNA damage, which may allow time for adequate DNA repair. We asked whether p53 could directly increase the repair of defined double-strand breaks (DSBs) by nonhomologous end-joining in gamma-irradiated mouse embryonic fibroblasts with differing p53 status. By using an episomal plasmid reactivation assay, we found that presence of wild-type p53 enhanced rejoining of DSBs with short complementary ends of single-stranded DNA. p53 appeared to be directly involved in this regulation, because rejoining enhancement was dependent on the presence of nonspecific DNA binding activity as mediated by the COOH-terminal domain and was independent of transactivating function. We hypothesize that tumor cells lacking p53 and normal cells with wild-type p53 may use different pathways for repair of radiation-induced DSBs.

High-dose radiation therapy alone for inoperable non-small cell lung cancer--experience with prolonged overall treatment times.

The purpose of this study as to determine the impact of overall treatment time on long-term survival after high-dose radiation therapy alone for inoperable non-small cell lung cancer (NSCLC). Between 1978 and 1990, 229 patients with stage I-III disease and Karnofsky Performance Scores of 80-100 received a conventionally fractionated total dose of 70 Gy through a split-course technique. After a first treatment course of 40 or 50 Gy, a restaging was performed and only patients without any contraindications, such as newly diagnosed distant metastases or serious deterioration of performance status, were given a second course. In 83% of patients this break lasted for 4-6 weeks. Overall treatment time ranged between 7 and 24 weeks (median 12 weeks). Median follow-up time was 6.6 years (range 4.0-9.3 years). Actuarial overall survival rates at 2 and 5 years were 28% and 7% respectively. Complete radiological tumor response was observed in 31% of patients, and was found to be the strongest positive predictor of survival with 2- and 5-year rates of 50% and 12% respectively compared with 17% and 4% for patients without complete response. Treatment duration was not found to be a significant prognostic factor in univariate or multivariate analysis. For overall treatment times of 7-11 weeks (n = 50), 12 weeks (n = 79) and > 12 weeks (n = 100), 5-year survival was 4%, 6%, and 8%, respectively (p = 0.6). To conclude, in our experience and in contrast to other studies, prolonged overall treatment times in radiation therapy alone for inoperable NSCLC had no negative impact on long-term survival. It is hypothesized that accelerated tumor cell repopulation is absent in a significant number of these patients with the time-factor playing no apparent role for outcome of treatment.

[One hundred years of radiotherapy. Historical origins and development of fractionated irradiation in German speaking countries]. / Ein Jahrhundert Strahlentherapie. Geschichtliche Ursprünge und Entwicklung der fraktionierten Bestrahlung im deutschsprachigen Raum.

This review studies the historical development of fractionated X-ray therapy in the German speaking countries until World War II. Fractionated treatments appear to have their origin already in the first attempts of fractionation performed by Freund in 1896. In the following, fractionated treatments could not compete with the so-called single dose or intensive radiation treatment in Germany. However, until the 1920s there were repeated sporadic attempts of various motifs to distribute the radiation doses over a prolonged period of time. Only with the end of the 1920s the conditions for a rise of fractionated irradiation were favourable, mainly due the fiasco of the intensive treatments and their subsequent decline. The impulse for this rise, however, came from France where Coutard had developed an individual empirical treatment regimen with great clinical success. This technique and its modifications and developments spread over Europe and America fast. In spite of the similarities between the various fractionation methods used during the first decades of the century, review of the radiological literature of that time fails to show any logical scientific connection of these methods, but rather there was a mix and overlap of irradiation attempts of various motivations. Thus, radiation therapy, and thereby the current standard fractionation scheme of 1.8 to 2 Gy per fraction 5 times per week, obviously did not grow out of a scientific basis, but originated from individual observation of patients and empirical experience.

[Proliferation rate and radiosensitivity. Bergonié's and Tribondeau's error]. / Proliferationsrate and Strahlenempfindlichkeit. Der Irrtum von Bergonié und Tribondeau.

In 1906 Bergonié and Tribondeau postulated a correlation between proliferation rate and radiosensitivity. Allowing for modern radiotherapeutic and radiobiological experience this postulate is no longer tenable.

Hyperfractionation: where do we stand?

Hyperfractionation is generally expected to allow an escalation of total dose, thereby increasing tumour control rate, without increasing the risk of late complications. The purpose of this review is to assess the empirical evidence for this therapeutic gain from hyperfractionated radiotherapy. Although extensive clinical data have been accumulated until now, especially on treatment of head and neck cancer, the line of evidence is not consistent. The present analysis indicates that the dose per fraction generally used in standard radiotherapy is already a good choice.

Impact of the interfraction interval on clonogenic cell survival in split-dose irradiation of R1H rhabdomyosarcoma of the rat in vitro.

PURPOSE: In a previous study, the response of the R1H rhabdomyosarcoma of the rat to conventional irradiation (1.83-2.75 Gy fractions once-daily) and hyperfractionated radiotherapy (0.92-1.38 Gy fractions with different time intervals between the two daily fractions) was investigated [Kleineidam, M., Pieconka, A., Beck-Bornholdt, H.-P. Radiotherapy of the rhabdomyosarcoma R1H of the rat: influence of the time interval between two daily fractions during hyperfractionated radiotherapy. Radiother. Oncol. 30: 128-132, 1994]. Compared to once-daily irradiation, interfraction intervals of 2 h led to reduced tumour response, due to recovery from sublethal damage, and intervals of 5-6 h resulted in increased tumour response, possibly due to cell cycle effects. The purpose of the present study was to complement these tumour data by measuring clonogenic cell survival of R1H cells in vitro after split-dose irradiation. METHODS AND MATERIALS: Experiments were performed with 2 x 2.5 Gy and 2 x 3.5 Gy either at 21 degrees C (preventing cell cycle progression) or at 37 degrees C (allowing for cell cycle effects). RESULTS: For 3.5 Gy fractions, a cell survival curve equivalent to the in vivo results was obtained with the lowest surviving fraction observed at time intervals of 6-7 h, but only when cells were incubated at 37 degrees C during the interval. This phenomenon was absent in the 21 degrees C experiments. CONCLUSIONS: Our data provide further evidence to support the hypothesis that cell cycle effects are responsible for such observations. We conclude that the length of the interfraction interval has a considerable potential effect on tumour response to altered fractionation regimens.

[Soft tissue sarcoma of the head and neck area in adults. Outcome of combined surgery and irradiation and radiotherapy alone]. / Weichteilsarkome des Kopf-Hals-Bereichs beim Erwachsenen. Ergebnisse der kombinierten Operation und Bestrahlung und der alleinigen Strahlentherapie.

PURPOSE: To analyse the experience treating soft tissue sarcomas of the head and neck at the Massachusetts General Hospital, Boston. Detailed results have been published previously [17]. PATIENTS AND METHOD: Between 1972 and 1993, 57 patients were treated curatively with radiation alone (n = 13) or combined surgery and pre- and/or postoperative irradiation (n = 44). Gross complete resection was achieved in 82% of patients and margins were negative in 5 patients. Doses ranged from 36.0 to 79.2 Gy (median 64.8 Gy), usually conventionally fractionated. In 16 patients protons were used. Median follow-up time was 4.3 years (range 1.1 to 16.8 years). RESULTS: After 5 years, patients with angiosarcomas (n = 11) and patients with other tumor types (n = 46) had locoregional control rates of 24% and 69%, distant failure rates of 58% and 17%, and overall survival rates of (for overall survival) and T stage (for locoregional control) (p < 0.05). Particularly, gross completely resected T1 tumors had a locoregional control rate of 91%. Patients with locoregional recurrence were at an increased risk to die (p = 0.004 in multivariate analysis). Patients with and without direct tumor extension to neurovascular structures, bones, organs, or skin had distant failure rates of 27% and 0%, respectively (p = 0.031). In multivariate analysis, direct extension was additionally a negative prognosticator of overall survival (p = 0.034). CONCLUSION: 1. Angiosarcomas of the head and neck have a considerably poorer prognosis than other soft tissue sarcomas of this region. 2. Head and neck sarcomas have a higher local recurrence rate than for example soft tissue sarcomas of the extremities. Optimisation of local treatment through combination of surgery and high-dose irradiation, however, can achieve improved results, especially for prognostically favourable subgroups. 3. In addition to tumor grade and size, direct tumor extension may be a useful additional staging parameter.

[Combined breast-preserving surgery, chemotherapy and radiotherapy in the treatment of breast carcinoma. Effect of the interval between surgery and the beginning of radiotherapy]. / Kombinierte brusterhaltende Chirurgie, Chemotherapie und Strahlentherapie bei der Behandlung des Mammakarzinoms. Der Einfluss des Intervalls zwischen Operation und Bestrahlungsbeginn.

BACKGROUND: The timing of breast conserving surgery, chemotherapy, and radiotherapy in breast cancer treatment has become the subject of increasing interest over the last years. PATIENTS AND METHOD: Seventy-four patients who underwent postoperative radiotherapy at our institution between 1985 and 1992 form the basis of this study. Median follow-up time was 5 years. Seventy-three percent of patients were pre- or perimenopausal. Almost all patients (91%) were UICC-stage II. Axillary lymph nodes were positive in 95% of cases. Complete gross resection was achieved in all patients, and in 65% final pathological margins were free of invasive or intraductal carcinoma. Postoperatively, 70% of patients received 6 cycles of polychemotherapy (predominantly CMF) before onset of irradiation. The radiation dose was in almost all cases 60 Gy including 10 Gy boost. RESULTS: Five years after start of treatment overall survival, disease-free survival, and local recurrence rates were 86% (95%-confidence limits, 76 to 93%), 73% (61 to 83%), and 8% (3 to 16%), respectively. For disease-free survival, the only significant prognostic factor was the number of involved lymph nodes: 0 to 3 = 86%, > or = 4 = 40% (p < 0.0001). The interval between surgery and radiation (< or = versus > 20 weeks) had no significant influence on disease-free survival or local tumor control. In contrast, there was a trend of increased regional and distant failure with shortening of the interval due to the delivery of less than 6 cycles chemotherapy before the onset of radiotherapy. CONCLUSIONS: In our experience, there was no negative impact of a delay of radiotherapy in order to deliver full course chemotherapy before initiation of radiotherapy. However, the low statistical power of this analysis due to the small number of patients must be considered. it appears possible that a less intense chemotherapy before starting radiation treatment correlates with enhanced distant failure and subsequently decreased disease-free survival rates. Therefore, for patients at increased risk for distant metastasis, we prefer to give 6 cycles polychemotherapy before irradiation.

[Historical experiences in trying different overall treatment times in x-ray therapy of malignant head and neck tumors in the 1920s and 1930s]. / Einige historische Erfahrungen mit der Erprobung unterschiedlich langer Gesamtbehandlungszeiten bei der Röntgentherapie maligner Kopf-Hals-Tumoren aus den zwanziger und dreissiger Jahren.

UNLABELLED: BACKGROUND/MATERIAL: Today as well as in the first decades of this century the time factor has been one central subject of radio-oncological research. Before World War II, all possible overall treatment times ranging from 1 day to several months were under clinical investigation. A major portion of these historical irradiation schemes is documented in the German written literature of this time period, particularly in the journal "Strahlentherapie". RESULTS: In Paris, Coutard continuously extended treatment duration in X-ray therapy of malignant head and neck tumors. As illustrated by a reevaluation of these treatment results, with his work Coutard has laid the first foundation for today's international widely used conventional overall treatment times of 6 to 7 weeks. Other authors in the 1920s and 1930s, like Zuppinger or Holthusen, preferred use of slightly shorter overall times. CONCLUSIONS: In retrospect, evidence from these historical evaluations must be regarded as very limited especially from a statistical standpoint of view. Without knowledge of the underlying raw data, particularly the results and conclusions of Coutard which had major impact on the later development of radiation therapy cannot be verified anymore. This indicates the value of publishing raw data in modern radio-oncology as well.