Clinical phosphoproteomic profiling for personalized targeted medicine using reverse phase protein microarray

Healthcare providers are increasingly incorporating information gleaned from genomics and proteomics in the diagnosis and treatment of cancer. These lines of inquiry are providing greater insight into why patients with similar histological tumor classification and staging often demonstrate dissimilar clinical outcomes, and are illuminating distinct diagnostic subgroups that are more responsive to specific treatment modalities. Clearer understanding of genes, gene products, and signaling pathways holds great promise for the personalization of molecular medicine. While the origins of oncologic disease are genetically encoded, the disease process is largely mediated through altered protein function. Recent investigations suggest that each individual patient’s tumor possesses unique kinase-driven cell signaling derangements, and that these derangements derive, in part, from the tumor’s relationship with its host microenvironment. Identification of signaling derangements and mapping functional protein–protein interactions via phosphoproteomic profiling offers great promise for the precise targeting of therapeutic agents, identifying new therapeutic targets, devising effective combinatorial therapies, monitoring treatment efficacy and toxicity, and ultimately predicting treatment outcome. This review focuses on advances in clinical phosphoproteomic profiling of cancer using the emerging technology of reverse phase protein microarrays, and highlights the translational roles this technology is playing in laying the foundations for personalized molecular therapeutics.     

Efficacy of low-dose dopamine infusion

Urine output following administration of a low-dose dopamine infusion was assessed in 20 very immature infants (median gestational age 27 weeks). Prior to the infusion, all infants had had a period of anuria. Urine output improved significantly during the second 24 h after commencing the infusion but, at that time period, urine output was greater than 2 ml/kg/h (designated a good response) in only 13 infants. There was no significant difference in gestational age, birth weight, period of anuria or fluid input of infants who had a good or a poor response to dopamine. Although the baseline blood pressure did not differ significantly between these two groups, the increase in blood pressure resulting from dopamine administration was significantly greater in those infants with a good response in urine output (p<0.02). We conclude that low-dose dopamine infusion can improve urine output in very immature infants. Our results suggest that there may be inter-individual variation in the sensitivity to dopamine.     

Molecular profiling of cancer

The objective of molecular profiling of cancer is to determine the differential expression of genes and proteins from human tissue in the progression from normal precursor tissue to preneoplastic tissue to cancer in order to discover diagnostic, prognostic, and therapeutic markers. With the development of high-throughput analytical techniques such as microarrays and 2-D PAGE as well as the development of tools for cell procurement from histological sections such as laser capture microdissection (LCM), it is now possible to perform molecular analyses on specific cell populations from tissue. Since recognition of specific cell populations is critical, there is a need to optimize fixation and embedding not only to improve preservation of biomolecules, but also to maintain excellent histology. We have shown that 70% ethanol fixation of prostate tissue improves the recovery of DNA, RNA, and proteins over routine formalin fixation and maintains histological quality comparable to formalin. There is also a need to develop new technologies in order to expand the range of tissue types that can be analyzed. The development and applications of Layered Expression Scanning (LES) for the molecular analysis of whole tissue sections are discussed.     

Three-phase radionuclide scintigraphy of the hand

Three-phase radionuclide scintigraphy of the hand was performed on 116 patients. Normal and abnormal patterns for radionuclide angiography, immediate post-injection blood-pool images, and delayed scans (3-4 hr.) were established. Of 80 patients with normal circulation, 61 (76%) had equal radial and ulnar artery flow bilaterally, while in 19 (24%) either the radial or ulnar artery was dominant. Abnormal studies were grouped into three categories: suspected vascular lesions (Group I), pain of uncertain etiology (Group II), and patients evaluated before and after reconstructive surgery (Group III). The diagnosis was correct in 89% of the patients in Group I (34/38), 89% of those in Group II (57/64), and all of those in Group III (14/14). Three-phase scintigraphy of the hand yields significant information about perfusion and bone metabolism.     

Differentiation of tumour-stage mycosis fungoides, psoriasis vulgaris and normal controls in a pilot study using serum proteomic analysis

BACKGROUND: Serum proteomic analysis is an analytical technique utilizing high-throughput mass spectrometry (MS) in order to assay thousands of serum proteins simultaneously. The resultant 'proteomic signature' has been used to differentiate benign and malignant diseases, enable disease prognosis, and monitor response to therapy. OBJECTIVES: This pilot study was designed to determine if serum protein patterns could be used to distinguish patients with tumour-stage mycosis fungoides (MF) from patients with a benign inflammatory skin condition (psoriasis) and/or subjects with healthy skin. METHODS: Serum was analysed from 45 patients with tumour-stage MF, 56 patients with psoriasis, and 47 controls using two MS platforms of differing resolution. An artificial intelligence-based classification model was constructed to predict the presence of the disease state based on the serum proteomic signature. RESULTS: Based on data from an independent testing set (14-16 subjects in each group), MF was distinguished from psoriasis with 78.6% (or 78.6%) sensitivity and 86.7% (or 93.8%) specificity, while sera from patients with psoriasis were distinguished from those of nonaffected controls with 86.7% (or 93.8%) sensitivity and 75.0% (or 76.9%) specificity (depending on the MS platform used). MF was distinguished from unaffected controls with 61.5% (or 71.4%) sensitivity and 91.7% (or 92.9%) specificity. In addition, a secondary survival analysis using 11 MS peaks identified significant survival differences between two MF groups (all P-values <0.05). CONCLUSIONS: Serum proteomics should be further investigated for its potential to identify patients with neoplastic skin disease and its ability to determine disease prognosis.     

Signal pathway profiling of epithelial and stromal compartments of colonic carcinoma reveals epithelial-mesenchymal transition

Molecular crosstalk, including reciprocal stimulation, is theorized to take place between epithelial cancer cells and surrounding non-neoplastic stromal cells. This is the rationale for stromal therapy, which could eliminate support of a cancer by its genetically stable stroma. Epithelial-stromal crosstalk is so far poorly documented in vivo, and cell cultures and animal experiments may not provide accurate models. The current study details stromal-epithelial signalling pathways in 35 human colon cancers, and compares them with matched normal tissues using quantitative proteomic microarrays. Lysates prepared from separately microdissected epithelium and stroma were analysed using antibodies against 61 cell signalling proteins, most of which recognize activated phospho-isoforms. Analyses using unsupervised and supervised statistical methods suggest that cell signalling pathway profiles in stroma and epithelium appear more similar to each other in tumours than in normal colon. This supports the concept that coordinated crosstalk occurs between epithelium and stroma in cancer and suggests epithelial-mesenchymal transition. Furthermore, the data herein suggest that it is driven by cell proliferation pathways and that, specifically, several key molecules within the mitogen-activated protein kinase pathway may play an important role. Given recent findings of epithelial-mesenchymal transition in therapy-resistant tumour epithelium, these findings could have therapeutic implications for colon cancer.     

Restoration of thrombolytic potential in plasminogen-deficient mice by bolus administration of plasminogen

Homozygous plasminogen-deficient (Plg-/-) mice had a significantly reduced thrombolytic capacity toward intravenously injected 125I-fibrin labeled plasma clots prepared from Plg-/- murine plasma (9% +/- 3% lysis after 8 hours; (mean +/- SEM, n = 6), as compared with 82% +/- 8% in wild-type mice; P < .0001). Bolus injection of 1 mg purified murine plasminogen in 10- to 17-week-old Plg-/- mice increased the plasminogen antigen and activity levels at 8 hours to normal levels (130 +/- 5 micrograms/mL). Plasminogen administration was associated with significant restoration of thrombolytic potential (64% +/- 7% spontaneous clot lysis; P < .0001 versus lysis without plasminogen injection). Bolus injection of 1 mg plasminogen in homozygous tissue- type plasminogen activator-deficient (t-PA-/-) mice doubled the plasminogen antigen and activity levels after 8 hours and increased 125I-fibrin clot lysis at 8 hours from 13% +/- 3% to 34% +/- 5% (P = .008). Fibrinogen, t-PA antigen and alpha 2-antiplasmin activity levels after 8 hours were not significantly different in the groups with or without plasminogen injection. Injection of plasminogen induced a variable increase (on average 7- to 10-fold) of PAI-1, but no correlation with the extent of spontaneous clot lysis was observed. Histopathologic examination at the end of the experiments revealed that fibrin deposition in the liver of Plg-/- mice was slightly reduced 8 hours after bolus plasminogen injection (P = .007) and markedly reduced after 24 hours (P < .0001). Plasminogen antigen levels in liver extracts were comparable with those found in wild-type mice at 8 hours (130 +/- 20 versus 110 +/- 15 ng/mg protein) and decreased to 25 +/- 3.2 ng/mg protein at 24 hours. Thus, restoration of normal plasminogen levels in Plg-/- mice normalized the thrombolytic potential toward experimentally induced pulmonary emboli, and resulted in removal of endogenous fibrin deposits within 24 hours.