O. John Semmes

Clinical proteomics: A need to define the field and to begin to set adequate standards

The aim of this manuscript is to initiate a constructive discussion about the definition of clinical proteomics, study requirements, pitfalls and (potential) use. Furthermore, we hope to stimulate proposals for the optimal use of future opportunities and seek unification of the approaches in clinical proteomic studies. We have outlined our collective views about the basic principles that should be considered in clinical proteomic studies, including sample selection, choice of technology and appropriate quality control, and the need for collaborative interdisciplinary efforts involving clinicians and scientists. Furthermore, we propose guidelines for the critical aspects that should be included in published reports. Our hope is that, as a result of stimulating discussion, a consensus will be reached amongst the scientific community leading to guidelines for the studies, similar to those already published for mass spectrometric sequencing data. We contend that clinical proteomics is not just a collection of studies dealing with analysis of clinical samples. Rather, the essence of clinical proteomics should be to address clinically relevant questions and to improve the state‐of‐the‐art, both in diagnosis and in therapy of diseases.

Tissue Imaging Using Nanospray Desorption Electrospray Ionization Mass Spectrometry

Ambient ionization imaging mass spectrometry is uniquely suited for detailed spatially resolved chemical characterization of biological samples in their native environment. However, the spatial resolution attainable using existing approaches is limited by the ion transfer efficiency from the ionization region into the mass spectrometer. Here, we present a first study of ambient imaging of biological samples using nanospray desorption ionization (nano-DESI). Nano-DESI is a new ambient pressure ionization technique that uses minute amounts of solvent confined between two capillaries comprising the nano-DESI probe and the solid analyte for controlled desorption of molecules present on the substrate followed by ionization through self-aspirating nanospray. We demonstrate highly sensitive spatially resolved analysis of tissue samples without sample preparation. Our first proof-of-principle experiments indicate the potential of nano-DESI for ambient imaging with a spatial resolution of better than 12 μm. The significant improvement of the spatial resolution offered by nano-DESI imaging combined with high detection efficiency will enable new imaging mass spectrometry applications in clinical diagnostics, drug discovery, molecular biology, and biochemistry.

Imaging Mass Spectrometry of a Specific Fragment of Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase Kinase 2 Discriminates Cancer from Uninvolved Prostate Tissue

Purpose: Histopathology is the standard approach for tissue diagnostics and centerpiece of pathology. Although the current system provides prognostic information, there is need for molecular markers that enhance diagnosis and better predict clinical prognosis. The ability to localize disease-specific molecular changes in biopsy tissue would help improve critical pathology decision making. Direct profiling of proteins from tissue using matrix-assisted laser desorption/ionization imaging mass spectrometry has the potential to supplement morphology with underlying molecular detail. Experimental Design: A discovery set of 11 prostate cancer (PCa)–containing and 10 benign prostate tissue sections was evaluated for protein expression differences. A separate validation set of 54 tissue sections (23 PCa and 31 benign) was used to verify the results. Cryosectioning was done to yield tissue sections analyzed by a pathologist to determine tissue morphology and mirror sections for imaging mass spectrometry. Spectra were acquired and the intensity of signals was plotted as a function of the location within the tissue. Results: An expression profile was found that discriminates between PCa and normal tissue. The overexpression of a single ion at m/z 4,355 was able to discriminate cancer from uninvolved tissue. Tandem mass spectrometry identified this marker as a fragment of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 2 (MEKK2). The ability of MEKK2 to discriminate tumor from normal cells was orthogonally confirmed. Conclusions: This study highlights the potential of this approach to uncover molecular detail that can be correlated with pathology decision making. In addition, the identification of MEKK2 shows the ability to discover proteins of relevance to PCa biology. (Clin Cancer Res 2009;15(17):5541–51)

A novel approach toward development of a rapid blood test for breast cancer

Mammography remains the diagnostic test of choice for breast cancer, but 20% of cancers still go undetected. Many serum biomarkers have been reported for breast cancer but none have proven to represent effective diagnostic strategies. ProteinChip mass spectrometry is an innovative technology that searches the proteome for differentially expressed proteins, allowing for the creation of a panel or profile of biomarkers. The objective of this study was to construct unique cancer-associated serum profiles that, combined with a classification algorithm, would enhance the detection of breast cancer Pretreatment serum samples from 134 female patients (45 with cancer, 42 with benign disease, 47 normal) were procured prospectively following institutional review board-approved protocols. Proteins were denatured, applied onto ProteinChip affinity surfaces, and subjected to surface enhanced laser desorption/ionization (SELDI) time-of-flight mass spectrometry. The SELDI output was analyzed using Biomarker Pattern Software to develop a classification tree based on group-specific protein profiles. The cross-validation analysis of cancer versus normal revealed sensitivity and specificity rates of 80% and 79%, and for cancer versus benign disease, 78% and 83%, respectively. When 2 different chip surfaces were combined the sensitivity and specificity increased to 90% and 93%, respectively. The sensitivity and specificity of this technique are comparable to those of mammography and, if confirmed in a larger study, this technique could provide the means toward development of a simple blood test to aid in the early detection of breast cancer. The combination of SELDI ProteinChip mass spectrometry and a classification- and regression-tree algorithm has the potential to use serum protein expression profiles for detection and diagnosis of breast cancer.

Human LINE-1 retrotransposon induces DNA damage and apoptosis in cancer cells

BackgroundLong interspersed nuclear elements (LINEs), Alu and endogenous retroviruses (ERVs) make up some 45% of human DNA. LINE-1 also called L1, is the most common family of non-LTR retrotransposons in the human genome and comprises about 17% of the genome. L1 elements require the integration into chromosomal target sites using L1-encoded endonuclease which creates staggering DNA breaks allowing the newly transposed L1 copies to integrate into the genome. L1 expression and retrotransposition in cancer cells might cause transcriptional deregulation, insertional mutations, DNA breaks, and an increased frequency of recombinations, contributing to genome instability. There is however little evidence on the mechanism of L1-induced genetic instability and its impact on cancer cell growth and proliferation.ResultsWe report that L1 has genome-destabilizing effects indicated by an accumulation of γ-H2AX foci, an early response to DNA strand breaks, in association with an abnormal cell cycle progression through a G2/M accumulation and an induction of apoptosis in breast cancer cells. In addition, we found that adjuvant L1 activation may lead to supra-additive killing when combined with radiation by enhancing the radiation lethality through induction of apoptosis that we have detected through Bax activation.ConclusionL1 retrotransposition is sensed as a DNA damaging event through the creation DNA breaks involving L1-encoded endonuclease. The apparent synergistic interaction between L1 activation and radiation can further be utilized for targeted induction of cancer cell death. Thus, the role of retrotransoposons in general, and of L1 in particular, in DNA damage and repair assumes larger significance both for the understanding of mutagenicity and, potentially, for the control of cell proliferation and apoptosis.

SELDI-TOF MS profiling of serum for detection of the progression of chronic hepatitis C to hepatocellular carcinoma†

Proteomic profiling of serum is an emerging technique to identify new biomarkers indicative of disease severity and progression. The objective of our study was to assess the use of surface‐enhanced laser desorption/ionization time‐of‐flight mass spectrometry (SELDI‐TOF MS) to identify multiple serum protein biomarkers for detection of liver disease progression to hepatocellular carcinoma (HCC). A cohort of 170 serum samples obtained from subjects in the United States with no liver disease (n = 39), liver diseases not associated with cirrhosis (n = 36), cirrhosis (n = 38), or HCC (n = 57) were applied to metal affinity protein chips for protein profiling by SELDI‐TOF MS. Across the four test groups, 38 differentially expressed proteins were used to generate multiple decision classification trees to distinguish the known disease states. Analysis of a subset of samples with only hepatitis C virus (HCV)‐related disease was emphasized. The serum protein profiles of control patients were readily distinguished from each HCV‐associated disease state. Two‐way comparisons of chronic hepatitis C, HCV cirrhosis, or HCV‐HCC versus healthy had a sensitivity/specificity range of 74% to 95%. For distinguishing chronic HCV from HCV‐HCC, a sensitivity of 61% and a specificity of 76% were obtained. However, when the values of known serum markers α fetoprotein, des‐gamma carboxyprothrombin, and GP73 were combined with the SELDI peak values, the sensitivity and specifity improved to 75% and 92%, respectively. In conclusion, SELDI‐TOF MS serum profiling is able to distinguish HCC from liver disease before cirrhosis as well as cirrhosis, especially in patients with HCV infection compared with other etiologies. (HEPATOLOGY 2005;41:634–642.)

Pharmacoproteomic analysis of prechemotherapy and postchemotherapy plasma samples from patients receiving neoadjuvant or adjuvant chemotherapy for breast carcinoma.

In this study, proteomic changes were examined in response to paclitaxel chemotherapy or 5‐fluorouracil, doxorubicin, and cyclophosphamide (FAC) chemotherapy in plasma from patients with Stage I–III breast carcinoma. The authors also compared the plasma profiles of patients with cancer with the plasma profiles of healthy women to identify breast carcinoma–associated protein markers.

National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines for Use of Tumor Markers in Clinical Practice: Quality Requirements

BACKGROUND This report presents updated National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines summarizing quality requirements for the use of tumor markers. METHODS One subcommittee developed guidelines for analytical quality relevant to serum and tissue-based tumor markers in current clinical practice. Two other subcommittees formulated recommendations particularly relevant to the developing technologies of microarrays and mass spectrometry. RESULTS Prerequisites for optimal use of tumor markers in routine practice include formulation of the correct clinical questions to ensure selection of the appropriate test, adherence to good clinical and laboratory practices (e.g., minimization of the risk of incorrect patient and/or specimen identification, tube type, or timing), use of internationally standardized and well-characterized methods, careful adherence to manufacturer instructions, and proactive and timely reactions to information derived from both internal QC and proficiency-testing specimens. Highly desirable procedures include those designed to minimize the risk of the reporting of erroneous results attributable to interferences such as heterophilic antibodies or hook effects, to facilitate the provision of informative clinical reports (e.g., cumulative and/or graphical reports, appropriately derived reference intervals, and interpretative comments), and when possible to integrate these reports with other patient information through electronic health records. Also mandatory is extensive validation encompassing all stages of analysis before introduction of new technologies such as microarrays and mass spectrometry. Provision of high-quality tumor marker services is facilitated by dialogue involving researchers, diagnostic companies, clinical and laboratory users, and regulatory agencies. CONCLUSIONS Implementation of these recommendations, adapted to local practice, should encourage optimization of the clinical use of tumor markers.

Serum Protein Profiles to Identify Head and Neck Cancer

Purpose: New and more consistent biomarkers of head and neck squamous cell carcinoma (HNSCC) are needed to improve early detection of disease and to monitor successful patient management. The purpose of this study was to determine whether a new proteomic technology could correctly identify protein expression profiles for cancer in patient serum samples. Experimental Design: Surface-enhanced laser desorption/ionization-time of flight-mass spectrometry ProteinChip system was used to screen for differentially expressed proteins in serum from 99 patients with HNSCC and 102 normal controls. Protein peak clustering and classification analyses of the surface-enhanced laser desorption/ionization spectral data were performed using the Biomarker Wizard and Biomarker Patterns software (version 3.0), respectively (Ciphergen Biosystems, Fremont, CA). Results: Several proteins, with masses ranging from 2,778 to 20,800 Da, were differentially expressed between HNSCC and the healthy controls. The serum protein expression profiles were used to develop and train a classification and regression tree algorithm, which reliably achieved a sensitivity of 83.3% and a specificity of 100% in discriminating HNSCC from normal controls. Conclusions: We propose that this technique has potential for the development of a screening test for the detection of HNSCC.

Computational protein biomarker prediction: a case study for prostate cancer

BackgroundRecent technological advances in mass spectrometry pose challenges in computational mathematics and statistics to process the mass spectral data into predictive models with clinical and biological significance. We discuss several classification-based approaches to finding protein biomarker candidates using protein profiles obtained via mass spectrometry, and we assess their statistical significance. Our overall goal is to implicate peaks that have a high likelihood of being biologically linked to a given disease state, and thus to narrow the search for biomarker candidates.ResultsThorough cross-validation studies and randomization tests are performed on a prostate cancer dataset with over 300 patients, obtained at the Eastern Virginia Medical School using SELDI-TOF mass spectrometry. We obtain average classification accuracies of 87% on a four-group classification problem using a two-stage linear SVM-based procedure and just 13 peaks, with other methods performing comparably.ConclusionsModern feature selection and classification methods are powerful techniques for both the identification of biomarker candidates and the related problem of building predictive models from protein mass spectrometric profiles. Cross-validation and randomization are essential tools that must be performed carefully in order not to bias the results unfairly. However, only a biological validation and identification of the underlying proteins will ultimately confirm the actual value and power of any computational predictions.