Robert E. Brown

Morphoproteomics combines the disciplines of histopathology, molecular biology and protein chemistry to paint a portrait of the protein circuitry in diseased cells for the purpose of uncovering molecular targets amenable to specific intervention, thereby customizing therapy for individual patients. This review considers the clinical application of morphoproteomics in malignant cells in the context of currently available pharmaceutical agents and discusses opportunities for combinatorial approaches that involve one or more small molecule inhibitors and single-agent chemotherapy with relatively low toxicity profiles. Future directions that involve focusing on points of convergence in signal transduction pathways and which integrate morphoproteomic with genomic and pharmacoproteomic and protein-function microarray data are offered.

Morphoproteomics utilizes immunohistochemistry to identify protein analytes in tumor cells in order to uncover or confirm potential molecular pathways that may be essential to their proliferation, integrity, and histogenesis and that may serve as therapeutic targets. This communication illustrates the application of such an approach to osteolytic Langerhans cell histiocytosis and considers such molecular targets in the context of currently available therapies.

Platelet-derived growth factor receptor-alpha (PDGFR-alpha) expression in the Hodgkin/Reed-Sternberg cells of nodular sclerosing Hodgkin's disease has been demonstrated in 2 studies. Additional receptor tyrosine kinases have also now been documented. This communication reports the correlative expression in nodular sclerosing Hodgkin's disease of activated (phosphorylated) p70S6K, one of the putative downstream effectors common to receptor tyrosine kinase signaling.

"Morphoproteomics" is defined as the identification by immunohistochemistry of the molecular circuitry of various proteins in a tumor by noting their state of activation (translocation and phosphorylation) and correlative expressions. This approach can uncover potential targets, amenable to therapeutic interventions, that are specific for an individual patient's tumor (ie, customized chemotherapy). This communication illustrates the successful application of such an approach in a patient with relapsed acute lymphoblastic leukemia.

The aim of this study was to elucidate protein circuitry in breast cancer based on the profiling of hormone-receptor-negative breast carcinomas using morphoproteomic and pharmacoproteomic techniques. Three human breast carcinoma cell lines (SKBR-3, MDA-175, MDA-231) were reacted by immunohistochemical (IHC) procedures to detect several categories of protein analytes. Immunoreactivities and cell compartmentalizations were scored from 0 to 3+ positivity using bright-field microscopy. An automated cellular imaging system (ACIS) was used to obtain a final combined score of staining intensity and positive cells in the IHC reactions, to enable comparisons with the visual scores and the rates of inhibition by pharmaceutical agents. FDA-approved inhibitors that target the protein circuitry were added to the cultures for 2-4 days. Proliferation assays were conducted, and in vitro inhibition rates were calculated as (control - treated)/control. Western blot analyses of whole cell lysates assessed the effects of the pharmaceutical agents on selected aspects of protein circuitry. Good to excellent correlation was observed between visual scores and ACIS scores (r values from 0.732 to 0.996 in 10 of 11 trials). Gleevec produced growth inhibition that correlated with the composite expressions of the platelet-derived growth factor (PDGF) family of ligands and receptors; captopril inhibited only MDA-175, consistent with its unique expression of plasmalemmal angiotensin-converting enzyme (ACE); and interferon (IFN)-alpha effected growth inhibition in accordance with the degree of conventional (c) protein kinase C (PKC)-alpha and phosphorylated (p)-PKCalpha/betaII expressions. Western blot analyses revealed correlative changes of several intracellular signals following incubation with these inhibitors. This study shows (a) a close association between the immunohistochemical expression of signal transduction markers and in vitro inhibition by pharmaceutical agents, and (b) correlations between the sites of action of the pharmaceutical agents and the downstream expression of proteins in hormone-receptor-negative breast cancer cell lines. Such morphoproteomic and pharmacoproteomic profiling of individual tumors may enable the pathologist and oncologist to design antitumor therapy that is customized for an individual patient.

Morphoproteomics is defined as the identification by immunohistochemistry of the molecular circuitry of various proteins in a tumor by noting their state of activation (translocation and phosphorylation) and correlative expressions. Such an approach can uncover or confirm potential molecular targets that may be essential to the growth, integrity, and histogenesis of a particular tumor type and that are amenable to specific therapeutic interventions. This communication illustrates the application of such an approach in depicting the mTOR pathway in mesenchymal chondrosarcoma.

This study characterizes 3 cases of mesenchymal chondrosarcoma (MC) utilizing a proteomic approach that allows for the detection, visual quantification, cellular compartmentalization, and assessment of the functional state of certain proteins that may promote tumor growth and/or oppose apoptosis. Immunohistochemical procedures were performed to detect the following protein antigens: CD99, interleukin (IL)-1alpha, IL-6, transforming growth factor (TGF)-alpha, conventional (c) protein kinase C (cPKC)-alpha, cPKC-betaII, phosphorylated (p)-PKC-alpha/betaII, c-kit (CD117), platelet-derived growth factor receptor (PDGFR)-alpha, PDGFR-beta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER)-2/neu, cathepsin D, angiotensin-converting enzyme (ACE), angiotensin II type 1 (AT1) receptor, p21ras, the alpha subunit of farnesyl and geranylgeranyl transferase (FTalpha/GGTalpha), phospho (p)-c-Jun N-terminal kinase (p-JNK), p-p38 mitogen-activated protein kinase (MAPK), cyclin D1, c-Jun, Ki-67, bcl-2, TGF-beta1 latency-associated peptide (LAP), TGF-betaRII, and cyclooxygenase (COX)-2. Immunoreactivities were scored from 0 to 3+ positivity using bright-field microscopy. The results showed that malignant mesenchymal chondroblasts exhibit stronger expressions of CD99, IL-1alpha, cPKC-alpha, p-PKC-alpha/betaII, PDGFR-alpha, p-JNK, Ki-67, and bcl-2 antigens than their more mature-appearing chondrocytic counterparts in MC. In conclusion, molecular profiling of mesenchymal chondrosarcoma using a proteomic approach characterized the mesenchymal chondroblasts as possessing pathways that incorporate PKC-alpha and PDGFR-alpha signaling and anti-apoptotic bcl-2 expression. Specific therapies to target the mesenchymal chondroblasts in mesenchymal chondrosarcoma might include interferon-alpha, rapamycin, ciprofloxacin, and STI571.

To identify molecular events that occur concomitantly in HER-2/neu protein-receptor-positive breast carcinoma and to identify pathogenetic and growth-modulating sequences around its tyrosine-kinase-mediated cell proliferation and tumorigenesis that may be amenable to therapeutic interventions. Slides containing sections of 3 pelleted human breast carcinoma cell lines (DAKO HercepTest) and expressing HER-2/neu protein-receptor scored at 3+ (SKBR-3), 1+ (MDA-175), and 0 (MDA-231), respectively, were reacted in immunohistochemical procedures for the detection of the following antigens: HER-2, estrogen receptor (ER), progesterone receptor (PR), Ki-67, cyclin D1, c-Jun, epidermal growth factor receptor (EGFR), transforming growth factor (TGF)-alpha, components of the JAK/STAT signal transduction pathway (gp130, interleukin [IL]-6, and IL-11), p21ras, farnesyl transferase (FT), and potential growth inhibitory/proapoptotic and antiapoptotic-related proteins (latency-associated peptide [LAP] of TGF-beta1, TGF-beta receptor [R] II, p53, and bcl-2 and cyclooxygenase [COX]-2). Immunoreactivities were graded using bright-field microscopy on a scale of 0 to 3+. Commonalities noted among the 3 cell lines include absent (0) chromogenic signals for ER and PR, relatively high Ki-67 proliferation indices (54, 40, and 61%, respectively), and positive signals (1 to 3+) for IL-6, IL-11, TGF-alpha, EGFR, TGF-beta1 (LAP), TGF-betaRII,FT, p21ras, and p53. Strong intranuclear immunopositivities for cyclin D1 and c-Jun antigens were evident in the MDA-231 cell line but absent or rare (0 to +/-) in SKBR-3. Conversely, gp130 antigen was readily detected in the SKBR-3 cell line but only weakly expressed (+/-) in MDA-231, whereas bcl-2 and COX-2 were expressed in the latter and not in SKBR-3 cells. These data suggest that signal transduction through farnesylated p21 ras is part of the pathogenesis of tyrosine-kinase-mediated proliferation in HER-2/neu protein-receptor-positive breast carcinoma. Collaborations with the EGFR and JAK/STAT systems in these molecular events are also likely. Potential therapeutic agents include downregulators of c-erb-B1 (EGFR) and -B2 (HER-2) receptor expressions, inhibitors of tyrosine kinase and farnesylation, and activators of growth inhibitory/proapoptotic pathways.