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.

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.

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.

Adult soft tissue sarcomas of the head and neck treated by radiation and surgery or radiation alone: patterns of failure and prognostic factors.

PURPOSE: To analyze our experience treating soft tissue sarcomas of the head and neck in adults, and to identify patterns of failure and prognostic factors. METHODS AND MATERIALS: The records of 57 patients with Stage M0 disease treated by radiation with or without surgery between 1972 and 1993 were reviewed. Median follow-up time was 4.3 years (range, 1.1-16.8 years). A group of potential prognostic factors was evaluated, including age at diagnosis, sex, initial tumor presentation (primary vs. recurrent), grade, T-stage, direct tumor extension, tumor depth, duration of treatment, and radiation dose. RESULTS: The subset of angiosarcomas (11 out of 57 patients) had a considerably adverse effect on treatment outcome for the total group of sarcomas, with actuarial 5-year overall survival (OS), locoregional control (LRC), and freedom from distant metastasis (FDM) rates being 31%, 24%, and 42%, respectively. In contrast, for the remaining 46 patients with other histopathological tumor types, OS, LRC, and FDM rates were significantly higher (74%, 69%, and 83%, respectively). For this group of patients, significant prognostic factors identified by uni- and multivariate analysis included tumor grade as a predictor of OS and T-stage as a predictor of LRC (p < or = 0.050). Those patients who experienced a locoregional recurrence were at a significantly increased risk of dying (p = 0.004 in a multivariate model). All 17 patients without direct tumor extension to neurovascular structures, bone, contiguous organs, or skin remained free from distant failure. In contrast, 27% of 29 patients with direct extension had developed distant metastases at 5 years. In multivariate analysis, the absence of direct extension was a positive predictor of FDM (p = 0.007) and of OS (p = 0.034). CONCLUSIONS: 1) Angiosarcomas of the head and neck have a considerably poorer prognosis than other soft tissue sarcomas of this site. 2) In addition to tumor grade and size, direct tumor extension may be a useful additional staging parameter. 3) High rates of locoregional failure in the head and neck area, a potential cause of morbidity and death, indicate a need for improved treatment strategies.

Origins of radiotherapy and radiobiology: separation of the influence of dose per fraction and overall treatment time on normal tissue damage by Reisner and Miescher in the 1930s.

The effect of fractionation on the response of normal tissues to irradiation was already investigated in the 1930s. Reisner (Reisner, A. Hauterythem und Röntgenstrahlung. Erg. Med. Strahlenforsch, 6: 1-60, 1933) measured the time course of skin erythema on thighs of humans by applying different doses per fraction while keeping constant total dose and overall treatment time. The results showed that acute skin damage was reduced with small doses per fraction. Two years later Miescher (Miescher, G. Tierexperimentelle Untersuchungen über den Einfluss der Fraktionierung auf den Späteffekt. Acta Radiol, 16: 25-38, 1935) published his results on late radiation effects in rabbit skin. He also reported that the main influencing factor for tissue tolerance was dose per fraction. In addition, he found indications that there was no impact of overall treatment time on the development of late reactions. Strandqvist in his famous monograph on the time factor in treatment of skin cancer (Strandqvist, M. Studien über die kumulative Wirkung der Röntgenstrahlen bei Fraktionierung. Erfahrungen aus dem Radiumhemmet an 280 Haut -und Lippenkarzinomen. Acta Radiol. (Suppl.) 55: 1-300, 1944), however, postulated that total dose and overall treatment time were the main determinants of local control as well as of normal tissue damage, apparently omitting to consider the findings of Reisner and Miescher in his own analysis. It is our impression that mainly due to the large influence of Strandqvist's work on radiobiological thinking the early findings on the normal tissue sparing effect of small fraction size have been forgotten and had to be rediscovered about 40 years later.

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.

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.

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.

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.

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.

Epidemiology of influenza in Lower Saxony during the period 1968-1978 with particular emphasis on subtypes A(H3N2) and A(H1N1) in winter 1977-78.

The influenza surveillance in Lower Saxony is based mainly on laboratory investigations, and especially those which involve the isolation of influenza viruses throughout the year. These investigations have provided information on the circulation of influenza viruses and of the antigenic drift during the Hong Kong period which resulted in many different variants. Since the advent of the Hong Kong virus subtype, a high excess mortality was observed only in the winter of 1969-70. In most of the other years there was a low excess mortality which coincided with the circulation of the influenza A viruses in the population. During the last winter 1978, influenza A viruses were not isolated before February. In February and March, the two distinct subtypes H3N2 and H1N1 circulated simultaneously. The influenza A(H1N1) virus attacked only children and young adults whereas the patients infected with influenza A(H3N2) virus ranged over all age groups.

[Epidemiology of non-A, non-B hepatitis]. / Epidemiologie der hepatitis non-A-non-B.

Hepatitis non-A-non-B is a nosologically homogeneous group of diseases probably caused by different infectious agents. Laboratory tests specific for these agents are not yet available. Epidemiologically one has to distinguish occurence of the disease after transfusion and sporadically occuring disease. In addition, epidemics have been reported, which seem to be similar to hepatitis A virus infection in regard to epidemiological and clinical pattern. Hepatitis non-A-non-B makes up for about 25% of all cases with hepatitis caused by virus, sporadically occuring cases predominate. In our own group of patients only 10% occured after transfusion. Hepatitis non-A-non-B occurs in adults, about equally in males and females; there are no saisonal influences to be observed. Blood transfusion, substitution of coalgulation factors, parenteral drug abuse and hemodialysis represent definite high risk situations. Nosocomial infections in professional people (doctors, nurses) and infections of sexual partners occure more rarely as is the case in hepatitis B. Perinatal transmission of the infectious agents of hepatitis non-A-non-B still has to be clarified.

[Seroepidemiology of acute infectious hepatitis (author's transl)]. / Sero-Epidemiologie der akuten Virushepatitis.

Hbs-Ag, anti-Hbs, anti-Hbc and anti-HA were determined and the concentration of IgM measured in the sera of cases of acute infectious hepatitis which occurred in the Hannover area in 1975. Although there was a high degree of contamination with hepatitis A virus among the population, acute infectious hepatitis A was rare (n = 56). The hepatitis A virus is principally transmitted by contact with infection or while traveling in southern Europe. The greatest part of infectious hepatitis is due to hepatitis virus B (n = 211). Non-A, non-B hepatitis was less frequently observed (n = 62). A high percentage of patients with serum hepatitis and non-A, non-B hepatitis gave a history of parenteral exposure to possibly infectious material.

[Improvement of rubella vaccination. Seroconversion after serial rubella vaccination with the jet injector]. / Zur Optimierung der Röteln-Impfung. Serokonversion nach Röteln-Reihenimpfung mit dem Jet-Injektor.

67.5% of 506 girls aged 12-19 years had antibodies against rubella. 168 seronegative girls were vaccinated with rubella vaccine Wistar RA 27/3, application by jet injector. All 166 serologically controlled girls developed HAI-antibodies with a geometric mean titer of 1:87. The geometric mean titer of the naturally immunized girls was 1:140.

[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.

[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.