WO2010102162A1 - Quantitation of serum cell signaling pathway proteins - Google Patents

Abstract

Methods are provided to concentrate and detect low abundance serum proteins, such as kinase and cell signaling pathway proteins using hydrogel nanoparticles to sequester and concentrate a protein of interest, and reverse phase protein microarrays to quantitate the protein The markers identified by the described methods have wide applicability in diagnosis and treatment of a variety of diseases including myocardial infarction, pulmonary embolus, stroke, and organ infarction

Description

Quantitation of Serum Cell Signaling Pathway Proteins

REFERENCE TO RELATED APPLICATIONS

This application claims an invention which was disclosed in Provisional Application Number 61/157,775, filed 03/05/2009, entitled "Quantitation of Serum Cell Signaling Pathway Proteins". The benefit under 35 USC § 119(e) of the United States provisional application is hereby claimed, and the aforementioned application is hereby incorporated herein by reference.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant No. DE-FC52-04NA25455, awarded by the Department of Energy. The government has certain rights in the invention. FIELD OF THE INVENTION

The invention pertains to the incorporation of hydrogel nanoparticles into a new method for concentrating and detecting low abundance serum proteins, such as kinase and phosphorylated cell signaling pathway proteins, that are very low in abundance, in order to promote early detection of diseases as well as gain insight into new therapies for the treatment of diseases. More particularly, the invention pertains to the use of hydrogel affinity bait nanoparticles capable of sequestering low molecular weight proteins, size sieving, target analyte sequestration, analyte concentration and protection from degradation prior to quantitation using reverse phase protein microarrays (RPMA). Hydrogel nanoparticles are defined as porous polymeric particles having diameter dimensions ranging from 1 nm to 10 μm.

BACKGROUND OF THE INVENTION

Current methods for discovering new serum protein biomarkers for the early diagnosis of disease do not have the sensitivity to detect low abundance proteins less than 10 nanograms per mL , particularly phosphoproteins and cell signaling proteins that underlie key signaling processes. Discovery and detection of serum protein biomarkers are hindered by severe physiologic challenges: a) the low abundance of serum biomarkers emanating from early stage tumors, b) the presence of high abundance proteins, such as albumin, that may interfere with detection of low abundant biomarkers, and c) degradation of the protein post collection (Liotta et al., 2003). Smart nanoparticles used to pre-process sera and plasma prior to RPMA analysis overcomes the numerous technical barriers to cell signaling biomarker discovery. The foremost problem in biomarker measurement is the extremely low abundance (concentration) of candidate markers in blood, which exist below the detection limits of mass spectrometry and conventional immunoassays. The second major problem is the overwhelming abundance of resident proteins (albumin and immunoglobulins), accounting for 90% of circulating plasma proteins, which confound and mask the isolation of rare biomarkers. A third serious challenge for biomarker measurement is the propensity for the low abundance biomarkers to be rapidly degraded by endogenous and exogenous proteinases immediately after the blood sample is drawn from the patient. When mixed with a biological sample, nanoparticles perform in one step, in-solution molecular size sieving chromatography and affinity chromatography. In addition proteins or peptides captured in the nanoparticles are completely protected from protease degradation. The present invention details a method that combines the preprocessing of sera with "smart" hydrogel nanoparticles with analysis by RPMA for identification and quantitative detection of low abundance candidate serum biomarkers. This method allows the improved ability to profile apoptosis, oxidative damage and proliferation/survival pathway proteins. This information may provide 1) early detection of disease, such as melanoma or cardiac damage, and 2) insights into new therapies or efficacy of therapies for disease treatment. SUMMARY OF THE INVENTION

Detection of low-abundance kinase and cell signaling protein biomarkers can provide information that can be useful in the diagnosis, treatment, and predicting a prognosis for disease. The methods of the invention are generally directed to sample processing using smart hydrogel nanoparticles to sequester and concentrate biomarkers of interest prior to analysis via RPMA. Thus, in a first aspect, the invention provides a method to pre-process bood samples allowing for the quantitative measurement of phosphorylation, cleavage, or total forms of kinases, phophatases, and other cell signaling proteins in serum samples that are undetectable by standard laboratory methods, such as ELISA and mass spectrometry. This technology permits the measurement of biomarkers at a level for which the disease is at an early stage and is highly treatable. In a second aspect, the multiplexed nature of the reverse phase protein microarray technology permits quantitative measurement of multiple cell signaling proteins. This method can be used to identify and test the efficacy of new therapeutic strategies formulated to cause cell death via cell cycle arrest, autophagy, and/or apoptosis or interference with microenvironment signaling. Finally, this combination of technologies can be used to validate potential biomarkers in serum and plasma that cannot be detected by non-antibody based methods, such as mass spectrometry.

BRIEF DESCRIPTION OF THE DRAWING

Fig. 1 shows serum pro-apoptotic protein Bak discriminates melanocytic lesions based on Breslow's thickness. A. Scatterplot of the relative intensity of Bak measured by RPMA in sera collected from patients with a Breslow thickness < 1 mm compared with patients with a Breslow thickness > 1 mm. The level of Bak was significantly lower (p < 0.03) in the first group. B. Scatterplot of relative intensity of PDGF-BB measured with RPMA in the sera collected from patients with a Breslow thickness > 1 mm compared with patients with a Breslow thickness < 1 mm.

Fig. 2 shows Spearman's rho non-parametric analysis for determining protein associations. Scatterplot Matrix of example signalling proteins measured with RPMA in sera of melanoma patients with a Breslow thickness > 1 mm and B. a Breslow thickness < 1 mm.

Fig. 3 shows Spearman's rho non-parametric analysis for determining protein associations. Scatterplot Matrix of example signalling proteins measured with RPMA in sera of melanoma patients with a Breslow thickness < 1 mm.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, it is to be understood that the embodiments of the invention herein described are merely illustrative of the application of the principles of the invention. Reference herein to details of the illustrated embodiments is not intended to limit the scope of the claims, which themselves recite those features regarded as essential to the invention.

Biomarker identification is hindered by a lack of sensitive, specific assays for detection, quantitation and validation of the proposed biomarker. The invention here presents a unique combination of nanoparticles sequestration and concentration of proteins, followed by an antibody based detection method (reverse phase protein microarrays), that provides a method for detecting and validating low abundance biomarkers in serum.

This invention can quantitatively measure the phosphorylation, cleavage, or total forms of kinases, phophatases, and other cell signaling proteins in serum samples that are undetectable by standard laboratory methods, such as ELISA and mass spectrometry. Early stage disease lesions will comprise a smaller volume and will elaborate lower concentrations of biomarkers. This technology permits the measurement of biomarkers at a level for which the disease is at an early stage and is highly treatable.

The multiplexed nature of the reverse phase protein microarray technology permits quantitative measurement of multiple cell signaling proteins. This work can be used to test the hypothesis that cell signaling activation portraits can predict a priori which targeted therapies will best cause cell death via cell cycle arrest, autophagy, and/or apoptosis or interference with microenvironment signaling.

This combination of technologies can be used to validate potential biomarkers in serum that have been detected by non-antibody based methods, such as mass spectrometry. The fabrication of kits containing essential enrichment and microarray reagents and materials would permit a practitioner to use the methods described herein, e.g., to identify a metabolic target in a cancer stem cell, to determine a personalized therapeutic regime, etc., also a feature of this invention. The kits can include hydrogel nanoparticles, a reverse phase protein microarray, reagents for use with a protein microarray, and/or the like. The kits can also include additional useful reagents, such as antibodies, buffers, and the like. Such kits also typically include, e.g., instructions for use of the compounds and other reagents, e.g., to practice the methods of the invention, as well as any packaging materials for packaging the components of the kits.

The included example describes the use of the technology to measure protein markers in the blood of patients with malignant melanoma. The markers identified in the apoptosis and prosurvival pathways have never been detected in the blood previously and have wide applicability to a variety of disease including myocardial infarction, pulmonary embolus, stroke, and organ infarction.

Introduction

Melanoma represents only 4% of all skin cancers, but nearly 80% of skin cancer deaths (Jemal et ai, 2008). Once melanoma spreads to regional and distant sites, the chance of cure decreases significantly. Unfortunately, current prognostic markers are often inadequate. The Breslow thickness remains the most powerful independent prognostic factor. However it doesn't truly address the complexity and heterogeneity of individual melanoma subtypes that can lead to success or failure of a targeted therapeutic agent. In fact, a minority of patients with thin melanomas will develop metastatic disease (Becker et ai., 2006).

Discovery of new serum protein biomarkers useful for early diagnosis and prognosis of cancer is an urgent goal of the field of proteomics ( Anderson and Anderson, 2002Ugurel et ai., 2009). Melanoma serum biomarkers are hindered by severe physiologic challenges: a) the low abundance of serum biomarkers emanating from a small dermatologic lesion, b) the presence of high abundance proteins, such as albumin, that may interfere with detection of low abundant biomarkers, and c) degradation of the protein post collection (Liotta et ai, 2003). A new class "smart" nanoparticles has been created to overcome these physiologic challenges. In this study, we employed core shell, bait-loaded nanoparticles that are capable of selectively entrapping low abundance and molecular weight target analytes and protecting them from enzymatic degradation (Fredolini et ai., 2008; Longo et ai., 2009; Luchini et ai., 2008)We used the nanoparticles to harvest serum proteins from patients with benign nevi, melanoma, or other non-melanoma lesions. In order to measure the candidate low abundance serum biomarkers with high sensitivity, the biomarkers captured by the nanoparticles were measured by another new technology: the Reverse Phase Protein Microarray (RPMA) (Espina et ai., 2003) platform. This combination of technologies permitted the successful measurement of activated signal pathway molecules that exist at extraordinary low concentrations in serum. We focused on apoptosis-related proteins due to the important role of apoptosis for growth regulation of neoplasms and particularly melanoma (Cory and Adams, 2002). Tchernev G, Orfanos CE. J Cutan Pathol. 2007 Mar;34(3):247-56. A third unique clinical research application applied in this study was in vivo reflectance mode confocal microscopy (RCM) (Pellacani et al., 2007). RCM was employed to morphologically characterize all melanocytic lesions before surgical excision and serum collection. The combination of smart nanoparticles, RPMA and confocal microscopy provide clinicians with the opportunity to correlate relevant morphologic aspects of melanocytic lesions with low abundance serum biomarker profiles.

In this example use of the subject invention we analyzed apoptotic pathway serum proteins from patients with benign nevi, melanoma, or other non-melanoma lesions combined with the lesion's in vivo morphologic aspects as an initial step in understanding the protein signature of individual melanoma subtypes in relation to their heterogeneous clinical and biologic behavior.

Results

Patient samples and clinical characteristics

The study population consisted of 29 serum samples from primary and metastatic melanoma (median age of 49.3 years, 37.8-58.3 interquartile range) and 26 serum samples from patients with atypical melanocytic nevi (median age of 54.5 years, 43.6-61.7 interquartile range) (Table 1). All sera were collected at University of Modena and Reggio Emilia, Italy under IRB approval with informed consent. Melanomas had a mean Breslow's thickness of 0.63 mm, 0.37-1.6 interquartile range. No in situ melanomas were reported; ten melanomas were equal or less than 1 mm (Tl staging according to AJCC) (Balch et al., 2001), five were between 1.01 and 2 mm (T2), three between 2.01 and 4 mm (T3), two thicker than 4 mm and three in transit metastasis. Atypical melanocytic nevi comprised 6 junctional nevi, 18 compound nevi, and 2 intradermal nevi.

Bak is differentially expressed in serum of melanoma patients compared to serum from patients with atypical nevi

To identify candidate biomarkers involved in early stage or progression of melanoma, serum collected from 29 patients with melanoma at different stages and from 26 individuals with atypical nevi were incubated with "smart" hydrogel nanoparticles (Luchini et al 2008, Longo et al 2009). The nanoparticles sequestered low abundance protein which were eluted for analysis by RPMA. A total of 24 signalling proteins were analyzed by RPMA. Each array was stained using antibodies against 18 phosphoproteins and other 6 signalling protein (Table 2). All 24 proteins were detected in sera of patients with melanoma and individuals with atypical nevi. This is the first evidence that phosphoproteins can be detected in serum following a new workflow that includes enrichment of low abundance proteins by means of serum preprocessing with NIPAm/AAc nanoparticles and subsequent analysis by RPMA.

Bak, a Bcl-2-related pro-apoptotic protein, was differentially abundant in serum collected from patients with melanoma compared to serum of individuals with atypical nevi

(sensitivity 79.9 % and specificity 80.8%, Bak threshold >3) by means of multivariate discriminant analysis.

To investigate the potential prognostic value of signalling proteins in serum we compared sera of patients with melanoma with a Breslow thickness < 1 mm with sera of patients with a Breslow thickness > 1 mm. Among the 24 end points analyzed, Bak was significantly lower (p < 0.03) in the sera collected from patients with a Breslow thickness > 1 mm compared with patients with a Breslow thickness < 1 mm (Figure 1 A). These results suggest that Bak may be a new candidate serum melanoma biomarker that could improve risk stratification of patients with melanoma. PDGF-BB, a very labile, low molecular weight protein present in serum at extremely low concentration was reliably measured by RPMA from NIPAm/AAc nanoparticle processed samples. PDGF-BB was not significantly different in the two subsets of analyzed sera (Figure 1 B).

Breslow's thickness is associated with serum prosurvival cell signalling pathway proteins

We grouped melanoma patients on the basis of their Breslow's thickness and we performed Spearman's rho non-parametric correlation analysis based on the level of the signaling proteins measured with RPMA. Serum from melanoma patients with Breslow's thickness greater than 1 mm showed significant correlation between prosurvival cell signalling pathway proteins mTOR Ser2481 and PDGF-BB whereas serum from melanoma patients with Breslow's thickness less than 1 mm showed significant correlation between apoptotic pathway proteins Cleaved Caspase 9 Asp315 and Bcl2 (Figures 2 and 3).

Correlation between Bak serum levels and in vivo melanoma imaging aspects

Confocal microscopy offers in vivo cellular-level resolution of lesion architecture. Bak serum levels measured by RPMA were correlated with previously identified relevant confocal parameters (Pellacani et al. , 2007). Within the evaluated confocal parameters, the presence of junctional nests and absence of sparse dermal nests, correlated with Bak serum levels greater than 4 relative intensity units. It is notable that all lesions showing junctional nests exclusively (and not dermal nests) were characterized by high Bak serum level (>4) whereas melanomas presenting sparse dermal nests on confocal images (in absence of junctional nests) had low levels of serum Bak (<4). The presence of both junctional and dermal nests, as well as absence of them, was not predictive of Bak serum levels (Table 3). No statistical significant differences were found in relation with cytologic aspects of melanomas as evaluated by RCM. A border line p value (p=0.086) was found for melanomas showing high Bak serum level and the collateral presence of dendritic shaped pagetoid cells and atypical melanocytes at the basal layer.

Correlation of Bak serum levels with IHC In order to confirm biologic relevant serum protein levels with traditional immunohistochemical techniques, IHC was performed on 13 melanoma cases. Positive IHC Bak scores (semi-quantitative scoring) were compared to Bak serum levels. Bak serum levels were significantly correlated with the presence of Bak in the epidermis (Spearman's rho 0.556, p=0.048). All cases with high Bak serum levels showed epidermal positivity for Bak by IHC analysis (data not shown) five of which were strongly positive. In contrast, cases with low Bak serum levels, only 1 case showed marked epidermal positivity for Bak and 2 cases exhibited weak immunoreactions; the remaining 2 cases were completely negative.

Discussion

Melanoma is the most devastating form of skin cancer and represents a leading cause of cancer death, particularly in young adults (Jemal et ai, 2008; Pellacani et al., 2008). Current morphological and histopathological criteria (anatomic site and type of the primary tumor, tumor size and invasion depth, ulceration, vascular invasion, and mitotic index) cannot truly address the complexity and heterogeneity of melanoma in relation to its unpredictable biologic behavior and prognosis. The heterogeneity of melanomas is manifested in a variety of clinical and biologic parameters. Distinct melanoma subtypes exist with specific growth rates (Liu et al., 2006). Moreover, melanoma differs based on body site location, age of onset, count of nevi, and skin phototype. This makes it difficult to combine all melanomas as a single clinical and biological entity. Examining the in vivo morphology of melanocytic lesions to reach a better clinical classification has been explored by using RCM. The availability of harvesting nanoparticle combined with the RPMA technology now makes it possible to discover and measure low abundance biomarkers that could not be detected in the past because they exited below the sensitivity of conventional assay methods. This example demonstrates that phosphoproteins can be detected in serum following a new workflow that includes enrichment of low abundance proteins by means of serum pre-processing with NIPAm/AAc hydrogel harvesting nanoparticles, with subsequent analysis by RPMA.

Hydrogel bait containing nanoparticles overcome the numerous technical barriers to biomarker discovery. The foremost problem in biomarker measurement is the extremely low abundance (concentration) of candidate markers in blood, which exist below the detection limits of mass spectrometry and conventional immunoassays. The second major problem is the overwhelming abundance of resident proteins (albumin and immunoglobulins), accounting for 90% of circulating plasma proteins, which confound and mask the isolation of rare biomarkers. A third serious challenge for biomarker measurement is the propensity for the low abundance biomarkers to be rapidly degraded by endogenous and exogenous proteinases immediately after the blood sample is drawn from the patient. We have previously demonstrated that hydrogel nanoparticles address all the major challenges associated with biomarker discovery and measurement. When mixed with a biological sample, nanoparticles perform in one step, in- solution molecular size sieving chromatography and affinity chromatography. In addition proteins or peptides captured in the nanoparticles are completely protected from protease degradation (Longo et al., 2009; Luchini et al., 2008). It has been previously shown that core shell nanoparticles composed of N-isopropylacrylamide-acrylic acid core (NIPAm/AAc) and a shell of N-isopropylacrylamide were able to concentrate PDGF-BB and other low abundant and short half-life chemokines (Longo et al. 2009). Moreover Luchini and co-workers demonstrated that NIPAm/AAc nanoparticles were suitable for capturing and concentrating low abundance biomarkers from serum for proteomic analysis (Luchini et al., 2008). The present study details the use of a new workflow that combines the pre-processing of sera with "smart" hydrogel nanoparticles with analysis by RPMA to identify low abundance candidate serum biomarkers. We demonstrate, for the first time, the detection of very low abundance pro-apoptotic proteins in sera from patients with melanoma and atypical nevi.

Programmed cell death is crucial for tissue homeostasis and for regulating physiological and pathological processes. Apoptosis is regulated by two key signaling pathways (extrinsic and intrinsic) that converge on the activation of caspase family proteins (cysteinyl aspartate proteases). The extrinsic pathway is regulated by the membrane death receptors, Tumor Necrosis Factor-receptor 1 (TNF-Rl) or Fas, that, when activated by their ligands, recruit adaptor proteins, such as FADD (Fas-associated death domain protein). Subsequently FADD recruits and activates caspase-8 that activates the downstream caspases, caspase-3 and -7, leading proteolysis of target molecules and consequent cell death (LA et ai, 2009). The intrinsic pathway, or mitochondrial pathway, is regulated by Bcl-2 family proteins and it is usually activated by cytotoxic events, such as cell stress or damage(Kang and Reynolds, 2009). The Bcl-2 family is composed of three classes of proteins: apoptosis inhibitors (such as Bcl-2, Bel- XL, BcI-W, McIl and BcI-B), pro-apoptotic proteins (Bax, Bak and Bok), and a third class that can regulate the activity of anti-apoptotic Bcl-2 proteins to induce apoptosis (Bad, Bid, Hrk,

BIM, Bmf, NOXA and PUMA). Pro-apoptotic Bax and Bak promote the release of cytochrome c or Diablo from mitochondria leading to caspase activation (Degli Esposti and Dive, 2003). On the other side, the anti-apoptotic Bcl-2 family proteins, such as Bcl-2 and BcI-XL, inhibit Bax and Bak (Youle and Strasser, 2008). The discovery of the Bcl-2 gene, its chromosome translocation, and its role in B-cell follicular lymphomas linked inhibition of apoptosis to cancer progression (Vaux, 1988 #233). Down regulation of apoptotic pathways which promote the survival of melanoma cells has been previously reported. The intrinsic apoptosis resistance of melanoma cells has been shown to be occur through over expression of pro-apoptotic proteins (Eberle et ai., 2003; Fecker et ai, 2006). In this present study, Bak levels were inversely correlated with melanoma thickness and cyto-architectural specific melanoma morphologies as observed by RCM.

Bak is a conserved homologue in the Bcl-2 protein family that acts at the mitochondrial membrane to facilitate release of cytochrome c, triggering caspase activation and apoptosis (Daniel et ai., 2003; Kirkin et ai., 2004). Its role as a negative prognostic factor (Fecker et ai., 2006) as well as its relevance in cisplatin-induced apoptosis has been reported (Mandic et ai.,

2001). In our study set, including melanoma and nevi, Bak was found to be a highly discriminatory protein between atypical melanocytic lesions and melanomas. Of note, the presence of high Bak serum levels was strongly correlated in melanoma containing junctional nests on confocal in vivo evaluation. These melanomas were traditionally classified as superficial spreading type. Among superficial spreading types, melanomas with junctional activity showed strong Bak positive immunoreactions in respect to those not having junctional nests. Taken together, these findings suggest that peculiar architectural and morphologic melanoma aspects, identified by RCM, have a differential Bak expression and a more intact apoptotic mechanism. Melanomas characterized by dermal nests, sparse type, and few to absent junctional activity, possessed low Bak serum levels. To our knowledge, no previous research studies have had the capability to link serum protein biomarker data with in vivo melanoma morphologies by means of dynamic RCM. The relevance of Bak expression in relation to melanoma morphologies holds the potential to understand the heterogeneous biologic lesions with high growth rate compared to others with indolent behavior. Measurement of BAK by immunohistochemistry correlated directly with the serum levels of BAK, and thereby provide a verification of this biomarker discovered using the subject invention.

The relationship between reflectance confocal microscopy and serum Bak levels was confirmed by IHC. Bak positive cells were mostly found in melanoma cases showing a higher

Bak serum level. These lesions were characterized by strong positive Bak immunoreactions for melanocytes located at dermo-epidermal junction and scattered through epidermal layers (pagetoid melanocytosis), sometimes forming small clusters. As melanocytes tended to form dermal aggregates, Bak positive cells decreased in number and weak Bak immunoreactivity was noted in the cytoplasm of cells in nests. It has to be emphasized that no in situ melanomas were included in our study population. This means that Bak is a potential novel melanoma biomarker that can discriminate melanomas along a continuum of Breslow's thickness, on the basis of slight morphologic differences.

Conclusions The present study demonstrates the clinical application of a novel biomarker harvesting nanotechnology that can be employed to discover candidate low abundance protein analytes in serum. Phosphorylated signal pathway proteins and other very low abundance proteins were successfully measured in serum. A regulator of apoptosis, Bak, was identified as a novel candidate prognostic serological biomarker for melanoma. This is in keeping with morphological and immunohistochemical observations performed on primary tumors from the same patients used in the serum study. Serum melanoma biomarkers could potentially improve risk stratification of melanoma patients.

Proteomic biomarkers for melanoma contain information not attainable by genomic markers. Thus a combination of proteomic and genomic/ genetic markers may offer the optimum means to stratify patients for individualized therapy. Materials and Methods Patient samples

55 sera were prospectively collected at the University of Modena and Reggio Emilia, Dept. of Dermatology, Modena, Italy under IRB approval (protocol number 1338/CE) with informed patient consent. The study population consisted of patients with melanocytic lesions excised following standard of care dermoscopic criteria for melanoma. All patients underwent venipuncture for serum collection. Lesions located on suitable sites were further characterized by in vivo confocal microscopy. All sera were collected before surgical excision— Sera were frozen in 600 μL aliquots at -8O⁰C without any additives. Smart nanoparticles were incubated with serum samples and the particle eluates were analyzed by RPMA.

Confocal microscopy image acquisition

Dermoscopic images were acquired using a digital dermoscope (3 Gen-Dermlite^®, LLC, San Juan Capistrano, CA) attached to a Konica Minolta Dimage Delta 10 camera. The RCM images were acquired by a near-infrared reflectance confocal laser scanning microscope (Vivascope 1500^®, Lucid Inc., Henrietta, New York). This system employs a diode laser at a wavelength of 830 nm and a 330 water immersion objective lens with numeric aperture of 0.9 (Rajadhyaksha et al., 1995). The laser power when it's applied to the skin is approximately 12 mW. RCM provides an in vivo lateral resolution of 2.0 μm, axial resolution of 5.0 μm, and an imaging depth of 250 μm. The RCM objective was attached to the skin via a steel ring, which in turn was attached to the epidermis with adhesive tape. A small drop of de-ionized water was used as immersion medium for the RCM objective. A dermoscopic image was acquired by means of VivaCam^® to provide spatial orientation during RCM acquisition, which provides correlation between the dermoscopic features and the confocal aspects. After dermoscopic image acquisition, a sequence of single confocal images and a montage of single high resolution images (500 μmx500μm), called "cube" images, were acquired, at the level of the epidermis, basal layer/dermo-epidermal junction and superficial dermis, respectively (Pellacani et al., 2005a). These mosaic images provide a lesion field of view that is currently up to 8x8 mm.

RCM features for 17 melanoma cases, were described by two expert observers (CL and GP), considering two major relevant confocal criteria : Architecture - i) presence of junctional nests, giving rise to a "meshwork pattern" that is represented by a predominance of junctional thickenings corresponding to enlargements of interpapillary space formed by aggregated cells and/or clusters bulging within dermal papilla in contiguity with basal layer (ii) presence of dermal nests, defined as "dense " dermal nests (compact aggregates of large polygonal cells) or "sparse" dermal nests (roundish nonreflecting structures with a well-demarcated border, containing bright nucleated cells), corresponding on histology to homogeneous or dishomogeneous clusters, respectively (Pellacani et al., 2007).

Cytology - presence of atypical melanocytic cells within the epidermis, distinguishing between a) cells spreading upwards in a pagetoid fashion and b) cells located at basal layer level. The shape of cells (roundish or dendritic) was evaluated (Pellacani et al., 2005b).

Core-shell particle synthesis and characterization

Core shell hydrogel nanoparticles were synthesized and characterized as previously described (Longo et al., 2009; Luchini et al., 2008). Briefly, nanoparticles were synthesized using N-isopropylacrylamide (NIPAm) (Sigma-Aldrich) and N, N' methylenebisacrylamide (BIS) (Sigma-Aldrich) by precipitation polymerization. Acrylic Acid (AAc) (Sigma-Aldrich) was incorporated into NIPAm particles to provide a charge based bait for affinity capture of peptides and small molecules.

Nanoparticles were washed to eliminate un-reacted monomer by subsequent centrifugations at 16.1 rcf, 25 ⁰C, 15 minutes. Supernatant was discarded and particles were re-suspended in 1 mL of water. The concentration of particles was assessed by weighing lyophilized particles and the count was performed by flow cytometry (BD). Particle size was evaluated by photon correlation spectroscopy (submicron particle size analyzer, Beckman Coulter).

Protein extraction/elution 500 μL of serum were diluted 1:3 with 50 mM Tris HCL pH 7 and incubated with 200 μL of nanoparticles for 15 minutes at room temperature. After incubation, samples were centrifuged for 15 minutes, 25 ⁰C at 16.1 rcf and supernatant was discarded. Then, the nanoparticles were re-suspended and washed in 1 mL of 20% acetonitrile-0.5x PBS and centrifuged for 7 minutes, 25 ⁰C at 16.1 rcf. Centrifugation and washing were repeated two times. A volume of 30 μL of 4X Laemmli Buffer was added to the nanoparticle pellet and the sample was boiled on a heating block at 100⁰C for 8 minutes. Samples were centrifuged (7 minutes, 25 ⁰C at 16.1 rcf) and the supernatant (nanoparticle elution) was saved and stored at -20 ° C before use. Prior to serial dilutions, samples were initially diluted 1:2 using a lysis buffer consisting of equal volumes of T-PER^® (Pierce, Rockford, IL, USA), and 2X Tris-glycine- SDS sample buffer (Invitrogen) in presence of 10% of TCEP (Thermo Fisher Scientific Inc.). The samples were heated on a heating block at 100⁰C for 5 minutes.

Construction of Reverse Phase Protein Microarrays (RPMA)

The nanoparticle eluates and control cell lysates were printed in duplicate on glass backed nitrocellulose array slides (FAST slides, Whatman) as previously described (VanMeter et al., MCP 2008). Briefly, reverse phase protein microarrays were printed using an Aushon 2470 arrayer equipped with 350 μm pins (Aushon Biosystem, Billerica, MA). Approximately 30 nL of each sample was printed in a dilution curve representing neat, 1 : 2, 1 :4 and 1 :8 dilutions. The slides were stored at -2O⁰C with desiccant (Drierite, W. A. Hammond, Xenia, OH) until use.

Immunostaining and analysis of reverse phase protein microarrays. RPMAs were blocked with PBS supplemented with 0.2% I-Block (Applied

Biosystems/Tropix) and 0.1% Tween 20 (Sigma-Aldrich) at least for 1 hour with constant rocking at room temperature.

RPMAs were stained as previously described (Espina et al. , 2003) with an Autostainer (Dako, Carpinteria, CA) following manufacturer directions using a catalyzed signal amplification system (Catalyzed Signal Amplification System; CSA) (Dako). Each slide was incubated at room temperature for 30 min with a single primary monoclonal or polyclonal antibody (Table 2). Subsequently each slide was incubated with a goat anti-rabbit IgG (H+L) (Vector Laboratories, Burlingame, CA) or rabbit anti-mouse IgG (1 : 10, Dako), depending on the source of the primary antibody used. Signal detection was performed using 3,3'-diaminobenzidine tetrahydrochloride (DAB, Dako). As a background control, one slide was selected for incubation with all reagents except for the primary antibody. All antibodies were subjected to extensive validation for single band, appropriate MW specificity by Western blot (Paweletz et al. , 2001). Stained arrays were scanned on a UMAX Powerlookll20 flatbed scanner (UMAX, Dallas, TX). Total protein content of each array spot was detected using Sypro Ruby Protein Blot Stain (Invitrogen, Carlsbad, CA) by a CCD equipped scanner (NovaRay, Alpha Innotech). The intensity of each spot was digitized using ImageQuant version 5.2 (GE Healthcare). The final relative mean intensity value was obtained by local background correction, subtracting the value obtained for each spot from the secondary antibody alone slide, and normalizing each spot to total protein. Immunohistochemical staining of tissue sections (IHC)

4 μm paraffin embedded sections were cut, mounted on adhesive-coated slides (Polylisine slides, Thermo Fisher, Rockford, IL USA) and dried at 6O⁰C for 30 min. One section from each patient was stained with hematoxylin and eosin, the remaining sections were stored at room temperature for immunohistochemical staining. Sections were dewaxed by heating at 6O⁰C and by two washes, 5 minutes each, with xylene. Tissue was rehydrated by a series of 5 minutes washes in 100%, 95% and 70% ethanol and water. Antigen retrieval was performed by heating the samples at 95⁰C for 30 minutes in 10mmol/L sodium citrate (pH 6.0). Endogenous activity was blocked with 3% hydrogen peroxide for 10 minutes followed by washing in distilled water for 5 min. After blocking with 2% albumin in Tris Buffered Saline with tween (TBST) buffer for 30 minutes, the slides were incubated with an antibody to Bak (1 : 500,

Epitomics) at 4⁰C overnight. After three washings in TBST for 5 min each, the sections were incubated with horseradish peroxidise anti-rabbit (1 : 500, Pierce) for 1 hour. After washing as before, the staining reaction was carried out with 100 μL of 3.3'-diaminobenzidine solution per slide (Sigma) for 5 min. The reaction was stopped with washing in water and then counterstained with Mayer's hematoxylin (10 min). The slides were dehydrated in a standard ethanol gradient, and sealed with coverslips. Negative controls were included by omitting Bak antibody during the primary incubation.

All tissue sample IHC slides were scored by two primary independent observers (FB, CL).

The positive reaction of Bak was scored into three grades according to the intensity of the staining : 0-absent, l-present in less than 50% of melanocytes, 2-present in more than 50

% of melanocytes. Two compartments were considered : Bak positivity within the epidermis and Bak positivity into the dermis (nests or single melanocytic cells).

Only S-100 positive cells were considered for Bak scoring. Images were acquired using an AxioCam MRc5 camera and Axioscope 40 microscope (Zeiss, Gδttingen, Germany). Statistical methods

Median and interquartile ranges were calculated for patient's age and melanoma Breslow's thickness.

RPMA data were quantified by ImageQuant software, and statistical analysis was performed with Excel, JMP and R statistical software. RPMA data were presented as the mean ± SEM. Student's t-test was used for statistical comparison between means where applicable.

Statistical evaluation was carried out employing the SPSS statistical package (release 12.0.0, 2003; SPSS Inc., Chicago, III., USA).

Non-parametric Spearman rho correlations between RPMA measured proteins and melanoma thickness were calculated (JMP ver5.2). Multivariate discriminant analysis was employed to determine independently significant proteins in order to predict melanoma diagnosis and to differentiate between thin and thick melanomas.

Relative and absolute frequencies of confocal features and immunohistochemistry patterns were evaluated in melanoma cases. Confocal features in melanomas, immunohistochemistry patterns and their correlation with indendently significant serum proteins were calculated with Spearman's rho test. Chi-square test of independence (Fisher's exact test was applied if any expected cell value in the 2x2 table was less than 5) was used to compare melanoma confocal and immunohistochemistry aspects with different Bak serum level.

References Anderson NL, Anderson NG (2002) The human plasma proteome: history, character, and diagnostic prospects. MoI Cell Proteomics 1 :845-867.

Balch CM, Buzaid AC, Soong SJ, Atkins MB, Cascinelli N, Coit DG, et al. (2001) Final version of the American Joint Committee on Cancer staging system for cutaneous melanoma. J Clin Oncol 19 : 3635-3648.

Becker D, Mihm MC, Hewitt SM, Sondak VK, Fountain JW, Thurin M (2006) Markers and tissue resources for melanoma : meeting report. Cancer Res 66: 10652-10657.

Cory S, Adams JM (2002) The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2:647-656. Daniel PT, Schulze-Osthoff K, Belka C, Guner D (2003) Guardians of cell death : the BcI-

2 family proteins. Essays Biochem 39 : 73-88.

Degli Esposti M, Dive C (2003) Mitochondrial membrane permeabilisation by Bax/Bak. Biochem Biophys Res Commun 304:455-461.

Eberle J, Fecker LF, Hossini AM, Wieder T, Daniel PT, Orfanos CE, et al. (2003) CD95/Fas signaling in human melanoma cells: conditional expression of CD95L/FasL overcomes the intrinsic apoptosis resistance of malignant melanoma and inhibits growth and progression of human melanoma xenotransplants. Oncogene 22:9131-9141.

Espina V, Mehta AI, Winters ME, Calvert V, Wulfkuhle J, Petricoin EF, 3rd, et al. (2003) Protein microarrays: molecular profiling technologies for clinical specimens. Proteomics 3 : 2091- 2100.

Fecker LF, Geilen CC, Tchernev G, Trefzer U, Assaf C, Kurbanov BM, et al. (2006) Loss of proapoptotic Bcl-2-related multidomain proteins in primary melanomas is associated with poor prognosis. J Invest Dermatol 126: 1366-1371.

Fredolini C, Meani F, Reeder KA, Rucker S, Patanarut A, Botterell P, et al. (2008) Concentration and Preservation of Very Low Abundance Biomarkers in Urine, such as Human

Growth Hormone (hGH), by Cibacron Blue F3G-A Loaded Hydrogel Particles. Nano Res 1 : 502- 518.

Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, et al. (2008) Cancer statistics, 2008. CA Cancer J Clin 58: 71-96. Kang MH, Reynolds CP (2009) Bcl-2 inhibitors: targeting mitochondrial apoptotic pathways in cancer therapy. CHn Cancer Res 15 : 1126-1132. Kirkin V, Joos S, Zornig M (2004) The role of Bcl-2 family members in tumorigenesis. Biochim Biophys Acta 1644: 229-249.

LA OR, Tai L, Lee L, Kruse EA, Grabow S, Fairlie WD, et al. (2009) Membrane-bound Fas ligand only is essential for Fas-induced apoptosis. Nature 461 :659-663. Liotta LA, Ferrari M, Petricoin E (2003) Clinical proteomics: written in blood. Nature

425:905.

Liu W, Dowling JP, Murray WK, McArthur GA, Thompson JF, Wolfe R, et al. (2006) Rate of growth in melanomas: characteristics and associations of rapidly growing melanomas. Arch Dermatol 142: 1551-1558. Longo C, Patanarut A, George T, Bishop B, Zhou W, Fredolini C, et al. (2009) Core-shell hydrogel particles harvest, concentrate and preserve labile low abundance biomarkers. PLoS ONE 4:e4763.

Luchini A, Geho DH, Bishop B, Tran D, Xia C, Dufour RL, et al. (2008) Smart hydrogel particles: biomarker harvesting : one-step affinity purification, size exclusion, and protection against degradation. Nano Lett 8 : 350-361.

Mandic A, Viktorsson K, Molin M, Akusjarvi G, Eguchi H, Hayashi SI, et al. (2001) Cisplatin induces the proapoptotic conformation of Bak in a deltaMEKKl-dependent manner. MoI Cell Biol 21 : 3684-3691.

Paweletz CP, Charboneau L, Bichsel VE, Simone NL, Chen T, Gillespie JW, et al. (2001) Reverse phase protein microarrays which capture disease progression show activation of pro- survival pathways at the cancer invasion front. Oncogene 20: 1981-1989.

Pellacani G, Cesinaro AM, Longo C, Grana C, Seidenari S (2005a) Microscopic in vivo description of cellular architecture of dermoscopic pigment network in nevi and melanomas. Arch Dermatol 141 : 147-154. Pellacani G, Cesinaro AM, Seidenari S (2005b) Reflectance-mode confocal microscopy for the in vivo characterization of pagetoid melanocytosis in melanomas and nevi. J Invest Dermatol 125: 532-537.

Pellacani G, Guitera P, Longo C, Avramidis M, Seidenari S, Menzies S (2007) The impact of in vivo reflectance confocal microscopy for the diagnostic accuracy of melanoma and equivocal melanocytic lesions. J Invest Dermatol 127: 2759-2765.

Pellacani G, Lo Scocco G, Vinceti M, Albertini G, Raccagni AA, Baldassari L, et al. (2008) Melanoma epidemic across the millennium : time trends of cutaneous melanoma in Emilia- Romagna (Italy) from 1997 to 2004. J Eur Acad Dermatol Venereol 22 : 213-218. Petricoin EF, Ardekani AM, Hitt BA, Levine PJ, Fusaro VA, Steinberg SM, et al. (2002) Use of proteomic patterns in serum to identify ovarian cancer. Lancet 359: 572-577.

Rajadhyaksha M, Grossman M, Esterowitz D, Webb RH, Anderson RR (1995) In vivo confocal scanning laser microscopy of human skin : melanin provides strong contrast. J Invest Dermatol 104:946-952.

Ugurel S, Utikal J, Becker JC (2009) Tumor biomarkers in melanoma. Cancer Control 16: 219-224.

Vaux DL, Cory S, Adams JM (1988) Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 335:440-442. Youle RJ, Strasser A (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev MoI Cell Biol 9:47-59.

Claims

CLAIMS:

We claim:

1 A method to concentrate and detect low abundance and labile cell signaling pathway analytes in biological fluids, comprising: hydrogel capture particles used to sequester and concentrate the analytes of interest; protein enrichment and isolation are conducted by particles ranging in size from lnm to lOOμm; containing an affinity bait internally within the particles; the particles comprising an open polymeric meshwork that encloses the affinity bait; recognizing the analyte via the affinity bait and the open polymeric meshwork; preventing degradation of the analyte inside the particles by concentrating and sequestering the analyte; quantification of sequestered and enriched analytes eluted directly from the particles using reverse phase protein microarray.

2 The method of claim 1, further comprising suspending the particles in the biological fluid containing the signaling analytes such that the particles that are suspended are of such buoyancy that the particles will remain in the sample fluid without settling.

3 The method of claim 2, further comprising maintaining the particles that are suspended such that the particles are of an open polymeric structure that is greater than 80 percent occupied by the sample fluid.

4 The method of claim 1, further comprising separating target analytes from the particle that are captured such that an extraction buffer is utilized to remove the analytes that are sequestered from the particles that are captured. 5 The method of claim 1, further comprising sequestering, concentrating and protecting proteins, peptides and nucleic acid analytes from degradation by contacting the sample fluid with the particles that are captured, the particles being a polymeric matrix having a pore size that allows for the analyte to enter the polymeric matrix while preventing other compounds located within the sample fluid to enter the polymeric matrix. 6 The method of claim 1, further comprising incorporating a population when the particles contain multiple subpopulations specific for individual classes of signaling analytes present in the sample fluid.

7 The method of claim 1, further comprising pre-concentrating and preserving the analyte when the analyte is present in the sample fluid and in the presence of hydrogel particles that contain the affinity bait for the analyte such that the hydrogel particles are of sufficient size and buoyancy to remain in the sample fluid, as well as when a high proportion of the analyte is sequestered within the hydrogel particles and a bulk of spent sample fluid volume is removed. 8 The method of claim 1, further comprising trapping the analyte within a capture particle, the capture particle formed by a molecular sieve portion and an analyte binding portion such that the molecular sieve portion and the analyte binding portion have a cross-linked region of a modified porosity while the molecular sieve portion is configured to contract and expand in response to physical and chemical treatment in order to trap the analyte within the capture particle.

9 The method of claim 1, further comprising eluting the captured analytes into an elution buffer whereas the elution buffer is capable of removing the trapped analytes for the capture particle affinity bait. 10 The method of claim 8, further comprising forming the analyte binding portion to be at least one type of moiety that is capable of chemically and electrostatically binding and sequestering the analyte.

11 The method of claim 8, further comprising forming the analyte binding portion to include a combination of a carboxy group, amine group, lipid, phosphoprotein, phospholipids, amide group, hydroxyl group, ester group, acrylic group, thiol group, acrylic acid, antibodies, binding proteins, binding pairs, metals, chelating agents, nucleic acids, aptamers, enzyme-binding pockets, lectins, pharmacologic agent, synthetic peptides, antibody fragments, hydrophobic surface, and a hydrophilic surface.

12 The method of claim 8, further comprising binding the analyte to the analyte binding portion such that the analyte is a combination of organic molecules, inorganic molecules, polypeptides, carbohydrates, nucleic acids, and lipids.

13 The method of claim 8, further comprising forming the molecular sieve portion to be an outer shell enclosing an inner core, the inner core made up of the analyte binding portion. 14 The method of claim 1, further comprising analyzing the captured analytes using

Reverse phase microarray technology such that the enriched analytes are spotted directly onto the microarray. 15 The method of claim 14, further comprising quantitation of the capture particle enriched analytes using reverse phase microarray technology. 16 The method of claim 14, further comprising quantitation of multiple analytes enriched from a single sample using capture particles for enrichment and reverse phase microarray technology for analysis. 17 A method of claim 13 further comprising the use of an immunoassay technology capable of measuring signal pathway proteins at a concentration less than 10 nanograms per mL.