Sabrina Heisel

Identification of lung cancer with high sensitivity and specificity by blood testing

BackgroundLung cancer is a very frequent and lethal tumor with an identifiable risk population. Cytological analysis and chest X-ray failed to reduce mortality, and CT screenings are still controversially discussed. Recent studies provided first evidence for the potential usefulness of autoantigens as markers for lung cancer.MethodsWe used extended panels of arrayed antigens and determined autoantibody signatures of sera from patients with different kinds of lung cancer, different common non-tumor lung pathologies, and controls without any lung disease by a newly developed computer aided image analysis procedure. The resulting signatures were classified using linear kernel Support Vector Machines and 10-fold cross-validation.ResultsThe novel approach allowed for discriminating lung cancer patients from controls without any lung disease with a specificity of 97.0%, a sensitivity of 97.9%, and an accuracy of 97.6%. The classification of stage IA/IB tumors and controls yielded a specificity of 97.6%, a sensitivity of 75.9%, and an accuracy of 92.9%. The discrimination of lung cancer patients from patients with non-tumor lung pathologies reached an accuracy of 88.5%.ConclusionWe were able to separate lung cancer patients from subjects without any lung disease with high accuracy. Furthermore, lung cancer patients could be seprated from patients with other non-tumor lung diseases. These results provide clear evidence that blood-based tests open new avenues for the early diagnosis of lung cancer.

Novel autoantigens immunogenic in COPD patients.

BackgroundChronic obstructive pulmonary disease (COPD) is a respiratory inflammatory condition with autoimmune features including IgG autoantibodies. In this study we analyze the complexity of the autoantibody response and reveal the nature of the antigens that are recognized by autoantibodies in COPD patients.MethodsAn array of 1827 gridded immunogenic peptide clones was established and screened with 17 sera of COPD patients and 60 healthy controls. Protein arrays were evaluated both by visual inspection and a recently developed computer aided image analysis technique. By this computer aided image analysis technique we computed the intensity values for each peptide clone and each serum and calculated the area under the receiver operator characteristics curve (AUC) for each clone and the separation COPD sera versus control sera.ResultsBy visual evaluation we detected 381 peptide clones that reacted with autoantibodies of COPD patients including 17 clones that reacted with more than 60% of the COPD sera and seven clones that reacted with more than 90% of the COPD sera. The comparison of COPD sera and controls by the automated image analysis system identified 212 peptide clones with informative AUC values. By in silico sequence analysis we found an enrichment of sequence motives previously associated with immunogenicity.ConclusionThe identification of a rather complex humoral immune response in COPD patients supports the idea of COPD as a disease with strong autoimmune features. The identification of novel immunogenic antigens is a first step towards a better understanding of the autoimmune component of COPD.

A Different View on DNA Amplifications Indicates Frequent, Highly Complex, and Stable Amplicons on 12q13-21 in Glioma

To further understand the biological significance of amplifications for glioma development and recurrencies, we characterized amplicon frequency and size in low-grade glioma and amplicon stability in vivo in recurring glioblastoma. We developed a 12q13-21 amplicon–specific genomic microarray and a bioinformatics amplification prediction tool to analyze amplicon frequency, size, and maintenance in 40 glioma samples including 16 glioblastoma, 10 anaplastic astrocytoma, 7 astrocytoma WHO grade 2, and 7 pilocytic astrocytoma. Whereas previous studies reported two amplified subregions, we found a more complex situation with many amplified subregions. Analyzing 40 glioma, we found that all analyzed glioblastoma and the majority of pilocytic astrocytoma, grade 2 astrocytoma, and anaplastic astrocytoma showed at least one amplified subregion, indicating a much higher amplification frequency than previously suggested. Amplifications in low-grade glioma were smaller in size and displayed clearly different distribution patterns than amplifications in glioblastoma. One glioblastoma and its recurrencies revealed an amplified subregion of 5 Mb that was stable for 6 years. Expression analysis of the amplified region revealed 10 overexpressed genes (i.e., KUB3, CTDSP2, CDK4, OS-9, DCTN2, RAB3IP, FRS2, GAS41, MDM2, and RAP1B) that were consistently overexpressed in all cases that carried this amplification. Our data indicate that amplifications on 12q13-21 (a) are more frequent than previously thought and present in low-grade tumors and (b) are maintained as extended regions over long periods of time. (Mol Cancer Res 2008;6(4):576–84)

Proteomic study of human glioblastoma multiforme tissue employing complementary two-dimensional liquid chromatography- and mass spectrometry-based approaches.

An extensive data set comprising 2660 unique protein identifications was obtained for the proteome of a human brain tumor (glioblastoma multiforme) by combining the results of two complementary analytical strategies based on two-dimensional chromatography and mass spectrometry. A bottom-up method, performing peptide separation in both chromatographic dimensions was employed as well as a semi-top-down method, in which intact proteins were separated in the first and tryptic peptides in the second dimension. The identified proteins were assigned to their molecular functions and compared to previously identified proteins of glioblastoma multiforme (= astrocytoma WHO grade IV), lower WHO grade astrocytomas (grade II and III), and nontumor brain tissue. With the use of a subset of 104 identified membrane proteins, the properties of intact protein fractionation in the first dimension of the semi-top-down approach were elucidated in detail. The benefit of the semi-top-down approach was further demonstrated by the identification of a set of endogenous glioblastoma multiforme expressed proteins. These proteins correspond to recombinant antigens which were recently found to be reactive against autoantibodies in glioblastoma multiforme patients. The results indicate the usefulness of the semi-top-down approach for the investigation of immunogenic antigens in human tumor tissue samples.

Novel immunogenic antigens increase classification accuracy in meningioma to 93.84

There is growing evidence that simultaneous analysis of multiple autoantibody reactions can be utilized for diagnosis of neoplasms. Using a set of 57 meningioma‐associated antigens, we recently separated meningioma patients from individuals without known disease with an accuracy of 90.3%. Here, we ask whether a largely increased set of immunogenic antigens can further improve this discrimination. We used an array with 1,827 human recombinant clones and measured reactivity of serum autoantibodies against the clones by a novel automated image analysis procedure. We were able to separate meningioma sera from sera of healthy controls with a specificity of 95.62%, a sensitivity of 91.83% and an accuracy of 93.84%. Of the analyzed clones, 23 in‐frame clones were highly informative for the classification of meningioma vs. normal sera as shown by their AUC values. These results demonstrate that the accuracy of a serum‐based diagnostic can be readily and considerably improved by screening extended sets of proteins.

The YEATS family member GAS41 interacts with the general transcription factor TFIIF.

BackgroundIn eukaryotes the transcription initiation by RNA polymerase II requires numerous general and regulatory factors including general transcription factors. The general transcription factor TFIIF controls the activity of the RNA polymerase II both at the initiation and elongation stages. The glioma amplified sequence 41 (GAS41) has been associated with TFIIF via its YEATS domain.ResultsUsing GST pull-down assays, we demonstrated that GAS41 binds to both, the small subunit (RAP30) and the large subunit (RAP74) of TFIIF in vitro. The in vivo interaction of GAS41 and endogenous RAP30 and RAP74 was confirmed by co-immunoprecipitation. GAS41 binds to two non-overlapping regions of the C-terminus of RAP30. There is also an ionic component to the binding between GAS41 and RAP30. There was no evidence for a direct interaction between GAS41 and TBP or between GAS41 and RNA polymerase II.ConclusionsOur results demonstrate binding between endogenous GAS41 and the endogenous TFIIF subunits (RAP30 and RAP74). Since we did not find evidence for a binding of GAS41 to TBP or RNA polymerase II, GAS41 seems to preferentially bind to TFIIF. GAS41 that does not contain a DNA-binding domain appears to be a co-factor of TFIIF.

GAS41 Amplification Results in Overexpression of a New Spindle Pole Protein

Amplification is a hallmark of many human tumors but the role of most amplified genes in human tumor development is not yet understood. Previously, we identified a frequently amplified gene in glioma termed glioma‐amplified sequence 41 (GAS41). Using the TCGA data portal and performing experiments on HeLa and TX3868, we analyzed the role of GAS41 amplification on GAS41 overexpression and the effect on the cell cycle. Here we show that GAS41 amplification is associated with overexpression in the majority of cases. Both induced and endogenous overexpression of GAS41 leads to an increase in multipolar spindles. We showed that GAS41 is specifically associated with pericentrosome material. As result of an increased GAS41 expression we found bipolar spindles with misaligned chromosomes. This number was even increased by a combined overexpression of GAS41 and a reduced expression of NuMA. We propose that GAS41 amplification may have an effect on the highly altered karyotype of glioblastoma via its role during spindle pole formation.

Large-scale antibody profiling of human blood sera: The future of molecular diagnosis

Despite the progress in cancer diagnosis the timely detection of many cancer types is still a grand challenge. For various human cancer types including lung cancer, prostate cancer, and breast cancer, several groups recently demonstrated that autoantibody profiling might be a promising approach towards earlier and more accurate cancer diagnosis.In this paper, we confirm the ability of autoantibody profiling as a diagnostic test by providing evidence that not only cancer sera can be distinguished well from normal controls, but also from sera of patients with noncancerous diseases. Altogether, we screened blood sera of 191 cancer patients, 60 physiologically unaffected controls, and 177 sera of patients with noncancerous diseases for more than 1800 immunogenic clones. The measured autoantibody fingerprints were evaluated using a novel image analysis pipeline.For 13 antigens, statistically significant (p<0.05) and at least two-fold elevated immuno-reactivity in cancer sera compared to normal sera could be observed. Nine of these antigens also showed increased reactivity compared to sera of patients with other diseases, including the tumor marker vimentin. Supervised discrimination between cancer and normal sera by using linear Support Vector Machines was possible with an accuracy of 94.04%, a specificity of 83.38%, and a sensitivity of 97.44%. Here, our so-called MIMM (minimally invasive multiple marker) approach showed no significant difference in the classification accuracy between low and higher tumor grades. The classification in healthy and diseased sera showed an even higher accuracy of 96.12% while the discrimination in cancer sera and diseased controls revealed an accuracy of 69.58%.These results demonstrate that autoantibody profiling offers the possibility of cancer screening for a variety of different cancer types as well as inflammatory diseases at an early disease stage.