WO2012019031A2 - Biomarkers for growth hormone disorders - Google Patents

Abstract

Disclosed embodiments describe certain protein isoforms and their relation to the progress of a treatment for a growth hormone (GH) disorder. Embodiments include methods of monitoring the efficacy of a treatment for a GH disorder in a subject, including: (a) detecting one or more protein isoforms of transthyretin, haptoglobin α2, beta-hemoglobin, apoA-I, and complement C4B precursor; and (b) determining altered levels of said markers, the altered levels indicating efficacy of the treatment. In addition, one or more of the above biomarkers may be used to assess the efficacy of a treatment for acromegaly. In alternative embodiments, one or more of the above biomarkers may be used to assess the efficacy of GH treatments to GH deficient patients. In yet other embodiments, one or more of the above biomarkers may be used to assess GH misuse.

Description

BIOMARKERS FOR GROWTH HORMONE DISORDERS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This non-provisional application claims the benefit of US Provisional Application No. 61/370,709 filed on 04 August 2010, the content of which is hereby incorporated by reference as if recited fully herein.

TECHNICAL FIELD

[0002] Embodiments relate to methods for assessing the efficacy of treatments for a GH disorder, including the detection of biomarkers.

BACKGROUND OF THE INVENTION

[0003] Growth hormone (GH) is synthesized and released by somatotrophic cells in the anterior lobe of the pituitary gland. Abnormal GH secretion leads to impairments in growth and metabolic processes. Acromegaly is an endocrine disorder, usually the consequence of a GH producing pituitary adenoma, characterized by elevated serum levels of GH and insulin-like growth factor- 1 (IGF-1). If left untreated, premature mortality ensues caused mainly from cardiovascular diseases. Current treatments include surgery; pharmaceuticals, such as pegvisomant; and or radiation therapy.

[0004] The primary treatment for acromegaly remains transsphenoidal surgery. GH secreting microadenomas are usually successfully removed by surgery, whereas the outcome is less favorable with larger tumors. Assessment of the surgical outcome is therefore important, with normalization of serum GH and IGF-1 levels as the current readout of successful treatment. However, these biomarkers are not optimal. Discrepant results of serum GH levels and IGF-1, i.e. elevated GH and normal IGF-1 or vice versa, are often found. Thus, the absence of reliable biochemical markers of acromegaly makes the evaluation of the successful treatment difficult.

SUMMARY OF THE GENERAL INVENTIVE CONCEPT

[0005] As embodied and broadly described herein, this disclosure relates to biomarkers for evaluating the efficacy of therapies or treatments for acromegaly, a disorder which results in elevated serum levels of GH and IGF- 1.

[0006] Embodiments include methods of monitoring the efficacy of a treatment for a GH disorder in a subject, comprising: (a) detecting one or more markers in a biological sample of the subject, said markers comprise a protein isoform selected from the group consisting of transthyretin (Swiss-Prot Acc. No. P02766) (SEQ ID NO. 1), haptoglobin a2 (Swiss-Prot Acc. No. P00738) (SEQ ID NO. 2), beta-hemoglobin (Swiss-Prot Acc. No. Q14484) (SEQ ID NO. 3), apoA-I (Swiss-Prot Acc. No. P02647) (SEQ ID NO. 4), and complement C4B precursor (Swiss-Prot Acc. No. P0C0L5) (SEQ ID NO. 5); and (b) determining altered levels of said markers, the altered levels indicating efficacy of the treatment. In various embodiments, one or more of the above biomarkers may be used to assess the efficacy of a treatment for acromegaly. In alternative embodiments, one or more of the above biomarkers may be used to assess the efficacy of GH treatments to GH deficient patients. In yet other embodiments, one or more of the above biomarkers may be used to assess GH misuse (e.g., athletes where GH is misused as a performance enhancing drug).

[0007] The above proteins are referred to herein as "marker proteins" or "biomarkers". The proteins can be used for confirming treatment efficacy by detecting changes in their concentration in the course of treatment for a disorder in which GH status is abnormal. In specific embodiments, specific protein isoforms in a subject's serum have diagnostic value for predicting the effectiveness of surgical treatment in acromegalic patients by detecting changes in their concentration in the course of treatment. Thus, embodiments also include a method of monitoring the effect of a treatment for acromegaly in a patient, which comprises detecting a change in concentration of at least one protein in a sample of a valid body tissue taken from the subject at a stage in said treatment, compared with the concentration of the protein in a sample of a valid body tissue taken from the subject prior to said treatment or at an earlier stage in the treatment, the protein selected from the group consisting of: transthyretin (Swiss-Prot Acc. No. P02766), haptoglobin 2a (Swiss-Prot Acc. No. P00738), beta-hemoglobin (Swiss-Prot Acc. No. Q14484), apoA-I (Swiss-Prot Acc. No. P02647), and complement C4B precursor (Swiss-Prot Acc. No. P0C0L5).

[0008] Accordingly, in various embodiments, the biological sample is subjected to two- dimensional gel electrophoresis to yield a stained gel and the increased or decreased concentration of the protein is detected by an increased or decreased intensity of a protein- containing spot on the stained gel, compared with a corresponding control gel (e.g., from a pre- treatment sample). In these embodiments, altered levels of spots A-G in FIG. 2 may be detected. In some embodiments, a decreased intensity of spot A, B, C, D, E, or F or an increased intensity of spot G in FIG. 2 in a post-treatment biological sample, as compared to the intensity of the spots in a pre-treatment sample, indicates efficacy of a surgical treatment for acromegaly. Hence, the biomarkers of the various embodiments may be defined alternatively in terms of the protein isoforms contained within the differentially expressed spots on a two dimensional electrophoretic gel, namely those identified as spots A-G in FIG. 2 herein, without regard to the names and database identifications given above. A biomarker of the various embodiments may be further defined in terms of its molecular weight (MW) and isoelectric point (PI), such that the protein isoform of spot A has a MW of about 15 and a pi of about 6.0; the protein isoform of spot B has a MW of about 15 and a pi of about 6.4; the protein isoform of spot C has a MW of about 20 and a pi of about 6.2; the protein isoform of spot D has a MW of about 12 and a pi of about 7.8; the protein isoform of spot E has a MW of about 10 and a pi of about 5.0; the protein isoform of spot F has a MW of about 10 and a pi of about 5.4; and the protein isoform of spot G has a MW of about 35 and a pi of about 7.5.

[0009] Another aspect of the embodiments includes a kit for monitoring the effect of a treatment for acromegaly in a subject, comprising ligands specific for protein isoforms comprising two or more of transthyretin (Swiss-Prot Acc. No. P02766), haptoglobin a2 (Swiss- Prot Acc. No. P00738), beta-hemoglobin (Swiss-Prot Acc. No. Q14484), apoA-I (Swiss-Prot Acc. No. P02647), and complement C4B precursor (Swiss-Prot Acc. No. P0C0L5). In some embodiments, the ligands comprise isoform specific antibodies.

[0010] Additional advantages of the disclosed method and compositions are in the description which follows, and in part are understood from the description, or may be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions are realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] A better understanding of the embodiments will be obtained from a reading of the following detailed description and the accompanying drawings in which:

[0012] Figure 1. Mean + SE serum levels of GH (A) andIGF-1 (B) at pre- (black) (5.29+2.14; 0.62+0.21) and post-treatment (white) (637+123.90; 242.25+40.95). *P<0.05. The P value is derived from two-tailed paired i-test. For statistics, please see text. GH (C) and IGF-1 (D) serum levels in individual subjects (1-8) pre- (black) and post-treatment (white).

[0013] Figure 2. Representative 2-DE gels showing their spot patterns. A total of 150 spots were analyzed (plus signs). Images belong to two different subjects (see labels) and correspond to the pre- and post-surgery treatment. Seven of those displayed significant intensity changes post-treatment (A-G; dashed (— ) squares). These protein spots were used for mass spectrometry identification.

[0014] Figure 3. (I) Plots showing intensity values (Mean + SE) before and after surgery for protein spots (A-G). (II) Spot intensity changes (intensity after treatment minus intensity before treatment) in individual subjects (1-8). Letters above each panel correspond to the spot shown in each graph. [0015] Figure 4. (I) Representative 2-D serum gel. The location of the seven protein spots displaying significant changes in intensity after transsphenoidal surgery are labeled A-G (— dashed squares). (II) Representative 3D view of protein spots A-G displaying intensities pre- treatment (left) and post- treatment (right). The images were generated using the 3D Viewer tool of PDQuest software version 8.0, which converts the spot intensity data to topographical peaks and valleys. For each protein spot, left and right images belong to the same subject.

[0016] Figure 5. A) Mean + SE serum levels of serum levels of haptoglobin pre- (white) (1.08 g/L +0.24) and post-treatment (black) (1.28 g/L +0.19). B) Western blots of haptoglobin, apoA- I and transthyretin in serum samples. Representative images of three different patients (ID: 1, 2 and 3) pre- (lanes 1, 3 and 5) and post-surgery (lanes 2, 4 and 6). Equal amounts of total protein were loaded (0.05 mg) and separated by SDS-PAGE.

[0017] Figure 6. Isoforms observable in 2-D western blots. A) 150 μg of total protein from serum was resolved in the 1^st dimension in a linear pH-range of 3-17 and in 15% acrylamide in the 2^nd dimension. (I) Representative 2-D gel showing the location of protein spots (A-C, E and F) (solid squares). Dashed (— ) squares show the MW and pi range of the different protein isoforms identified on 2-D western blots. Protein spots A and B were identified as transthyretin (1) (MW -15, pi -6.0; MW-15, pi -6.4). Protein spot C was identified as haptoglobin a2 (2) (MW -20, pi - 6.2). Isoforms of apoA-I (3) were identified at a MW -28 and a pi range of -5.2-7.8. Protein spots E and F (4) (MW - 10, pi -5.0; MW -10, pi -5.4) identified by MS as apoA-I (see table 1) were not detected by western blotting. Molecular weight markers are indicated to the left of the panels and pi markers are shown on the top. (II) Representative 2-D western blots showing the identified isoforms (— dashed squares) and the localization of protein spots A-C (solid squares).

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENT(S)

[0018] Biomarkers, as markers of therapeutic response, are essential for assessing the effectiveness of acromegaly therapies, such as transsphenoidal surgery. Currently, measurement of GH and IGF-1 levels is the standard assay for disease activity for assessment of disease activity in acromegaly. Unfortunately, these indicators are not reliable. Both GH and IGF-1 assays are subject to substantial variability and there is no consensus on assay standards. Moreover, cutoff values for GH and IGF-1 must ideally be corrected for age, gender, and body composition.

[0019] In various embodiments, two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to identify specific protein isoforms for monitoring and confirming the efficacy of a treatment for acromegaly. As described herein, differential expression of one or more isoforms from proteins including transthyretin (Swiss-Prot Acc. No. P02766; gil55669575), haptoglobin a2 (Swiss-Prot Acc. No. P00738; gil296653), beta- hemoglobin (Swiss-Prot Acc. No. Q14484; gil61679768), and apoA-I (Swiss-Prot Acc. No. P02647; gil90108664), or complement C4B precursor (Swiss-Prot Acc. No. P0C0L5; gil 1314244), was correlated with decreased GH and IGF-1 levels following successful transsphenoidal adenomectomy. Accordingly, the protein set identified and described herein comprises biomarkers useful to monitor and confirm the efficacy of acromegaly treatments such as transsphenoidal adenomectomy.

[0020] The Swiss-Prot accession number along with the "gi" number is provided for the various biomarkers. Sequence information relating to each biomarker is disclosed on The National Center for Biotechnology Information (NCBI) Protein database at http://www.ncbi.nih.gov/protein. The acronym GI stands for "Genlnfo Identifier." GI number (sometimes written in lower case, "gi") is a series of digits that are assigned consecutively to each sequence record processed by NCBI. The GI number has been used for many years by NCBI to track sequence histories in GenBank and the other sequence databases it maintains. Additionally, there are a variety of sequences related to biomarkers as well as any other protein disclosed herein that are disclosed on Uniprot (UniProtKB/Swiss-Prot), EMBL, DDBJ, and or Genbank databases, and these sequences and others are herein incorporated by reference in their entireties as well as for individual subsequences contained therein. Those of skill in the art understand how to resolve sequence discrepancies and differences and to adjust the compositions and methods relating to a particular sequence to other related sequences.

[0021] Disclosed herein are biomarkers and methods for identifying and using the biomarkers. By biomarker is meant any assayable characteristic or composition that is used to identify, predict, or monitor a condition (e.g., acromegaly; a disorder in which normal GH levels are elevated, or GH deficiency; a disorder in which GH levels are depressed) or a therapy for said condition in a subject or sample. A biomarker is, for example, a protein and or a particular protein isoform, or combination of proteins and or protein isoforms, whose presence, absence, or relative amount is used to identify a condition or status of a condition in a subject or sample. In at least one example, a biomarker is a protein isoform, or a combination of protein isoforms, whose relative concentration in a subject or sample is characteristic of a therapeutic reduction in serum GH levels.

[0022] Various embodiments include methods and materials for monitoring the effect of a treatment for a GH disorder in a subject. In various embodiments, methods include analyzing the serum proteome of acromegalic patients before and after successful transsphenoidal adenomectomy (or other surgical procedure). Seven serum proteins were found that significantly change following successful surgery in acromegalic patients. These proteins spots were identified as isoforms of transthyretin (MW -15, pi -6.0; MW-15, pi -6.4), haptoglobin 2a (MW -20, pi - 6.2), beta-hemoglobin (MW -12, pi - 7.8), apoA-I (MW -10, pi -5.0; MW-10, pi -5.4); and complement C4B precursor (MW -35, pi -7.5). These proteins represent reliable biomarkers of disease activity that is successfully mitigated by surgery.

[0023] Disclosed herein is the use of specific isoforms of transthyretin as a biomarker for predicting the effectiveness of a treatment for a GH disorder such as acromegaly (identified as spots A and B in FIG. 2). According to the Swiss-2DPAGE database, four isoforms of transthyretin (MW -13.8-35.39, isoelectric point (pi) -5.02-5.52) have been detected in human plasma by 2-DE. Recent reports have identified additional isoforms of this protein in mouse serum at different pis and MWs to those previously recorded in the databases. One possible explanation for the various isoforms is the presence of post-translational modifications that may lead to shifts in pi and/or MW. Thus, the differences in mass and/or charge of the isoforms observed in the reported samples may be due to these types of protein modifications, e.g., glycosylations and phosphorylations. Additionally, protein cleavage could be responsible for major changes in the mass of a given protein. To further support the MS results, conventional 1- D and 2-D western blots were performed. The 2-D pattern of transthyretin was very similar on the western blotting film compared to 2-D gels. 2-D immunoblots showed the presence of several transthyretin isoforms at two different MWs of -15 and -35 kDa and in pi range of -5.5-7.2. Two of these transthyretin isoforms corresponded to protein spots A and B (Spot A MW -15, pi -6.0; Spot B MW -15, pi -6.4), confirming the identity assigned by MS. Interestingly, no significant differences in total transthyretin levels were found by 1-D immunoblots, suggesting that quantifying total levels of transthyretin does not accurately reflect changes associated to the surgical outcome in acromegalic patients. One obvious limitation of conventional 1-D western blots is that it only provides information on the 'total' level of protein, but not on the differences in expression of particular isoforms of the target protein. In summary, although several isoforms of transthyretin are present in serum, the 2-D proteomic results clearly show that only two transthyretin isoforms (see FIG. 2, spots A and B) were significantly decreased in acromegalic patients after the surgical procedure. This may suggest that the enzymes responsible for the post-translational changes in transthyretin may be responding to GH action (or novel targets for GH).

[0024] Disclosed herein is the use of a specific isoform of haptoglobin a2 as a biomarker for predicting the effectiveness of a treatment for a GH disorder such as acromegaly (identified as spot C in FIG. 2). Haptoglobin (Hp) is an inflammation-inducible plasma protein which is present in the serum of all mammals, but polymorphism is only found in humans. The Hp molecule contains 2 different chains: β (heavy, 40 kDa) and a (light al, 8.9 kDa; a2, 16 kDa). In the mature protein, the a and β chains are connected by disulfide bridges (β- - -β). Two alleles (denoted 1 and 2) exist for the haptoglobin gene in humans. Therefore, 3 possible phenotypes results of these two alleles: Hp 1-1 ((α1β)2), Hp 2-2 ((α2β)3) and Hp 2-1 ((α1β)2, trimeric αβ or (αβ)3; where a represents a mixture of al and a2 chains) (38, 39). The β chain is present in all Hp phenotypes and it is always identical. Thus, Hp variations are due to the presence of different a chains. Interestingly, studies have shown association between Hp phenotype and disease. For example, one study showed that diabetic patients with Hp 2-2 present an increased risk to develop coronary heart disease than those with Hp 1-1. Additional studies have shown that Hp a2 chain expression is increased in patients with neck and head cancer. Twenty four isoforms (MW -11.86-44.46, pi -4.81- 6.07,) of haptoglobin have been identified by 2-DE in human plasma. Our data showed that the protein expression of one isoform of haptoglobin-2a (MW -20, pi -6.2) was markedly down-regulated after the surgical procedure. Interestingly, no significant changes in the total concentration of Hp were detected by a conventional ELISA assay. Supporting these results, 1-D western blot showed no significant differences in total Hp levels. These findings suggest that the evaluation of total serum Hp by conventional assays do not accurately reflect the changes after surgical treatment of acromegalic individuals. Several isoforms of haptoglobin-2a isoforms (MW -20, pi -5.7- 7.5) were identified by 2-D western blots. One of the isoforms corresponds to protein spot C (MW -20, pi -6.2), confirming our MS results.

[0025] Disclosed herein is the use of a specific isoform of beta-hemoglobin as a biomarker for predicting the effectiveness of a treatment for a GH disorder such as acromegaly (identified as spot D in FIG. 2). Previous studies have shown that total hemoglobin (Hb) concentration has been positively correlated with IGF-1 and IGFBP-3 levels. Studies have also implicated GH/IGF axis proteins as stimulatory factors for erythropoieisis, which results in increased Hb levels. However, these studies relate to heterotetrameric Hb in erythrocytes but not to the expression of free hemoglobin and/or specific hemoglobin isoforms. Serum free hemoglobin (alpha and beta chains) has been identified as potential biomarkers for ovarian and prostate cancer. In addition, a recent report by Chung et al. reported hemoglobin alpha-chain as a biomarker of GH in serum. Moreover, serum proteomic studies by Sackmann-Sala et al. identified a third isoform of beta-hemoglobin at a pl~8.0 with MW of -12 that changed due to increased GH/IGF-1 action. Two isoforms of beta-hemoglobin (MW -10.53, pi -6.88-7.05) are reported in the Swiss-2DPAGE database. In the current study, we found that the expression of one isoform of beta-hemoglobin (MW -12, pi -7.8) was decreased in the serum samples collected after surgery. We were initially concerned with the result since the presence of hemoglobin may be due to an artifact. However, this is unlikely since ail samples were handled consistently, and none of the samples were seemingly red due to red blood cell contamination. Moreover, we observed that only one isoform of free hemoglobin was differentially expressed following the pituitary surgery. No changes in additional isoforms were detected.

[0026] Disclosed herein is the use of a specific isoforms of apoA-I as a biomarker for predicting the effectiveness of a treatment for a GH disorder such as acromegaly (identified as spots E and F in FIG. 2). ApoA-I is a major component of high density lipoproteins (HDL) in human plasma and promotes cholesterol efflux from the tissues to the liver. In plasma, nine isoforms of apoA-I (pi -4.79-7.27, MW -7.49-23.45) have been reported. We have identified two isoforms of apoA-I (MW-10, pl~5.0 and MW-10, pl~5.4) that were significantly decreased post- surgical treatment. Studies have shown that GH plays a role in the modulation of lipid metabolism in humans. However, the effects of GH on cholesterol metabolism are still controversial. Some clinical studies on GH deficient children showed no significant effects on the levels of total apoA-I and HDL-cholesterol after GH replacement treatment, while others revealed decreases in apoA-I serum levels. In addition, normalization of GH serum levels in acromegaly patients after surgical and/or pharmacological treatment has been associated with increases in circulating apoA-I. In the present study, no significant differences in total apoA-I levels were found. As previously discussed, results presented herein do not contradict previous observations on the effect of GH in lipid metabolism, given that the decrease in the particular isoforms detected (E and F) does not reflect the total apoA-I levels in serum. Since there are several isoforms of apoA-I in blood as described above which may represent several post- translational modifications, the possibility exists that they may respond differently to GH. In addition, results presented herein reflect changes in the expression of two isoforms of apoA-I in response to the pituitary surgery, but not necessarily the correlation between GH activity and apoA-I levels.

[0027] To confirm the MS results for apoA-I, western blots were performed. Although, several isoforms of apoA-I were identified by 2-D western blots at a MW-28 kDa and a pi range of 5.2-7.8, apoA-I isoforms at lower MWs were not detected by this technique. A possible explanation is that post-translation modifications, e.g. proteolytic cleavages, associated to the apoA-I isoforms identified by MS may prevent the interactions between the antibody and its epitope.

[0028] Disclosed herein is the use of a specific isoform of complement C4B precursor as a biomarker for predicting the effectiveness of a treatment for a GH disorder such as acromegaly (identified as spot G in FIG. 2). An isoform of complement C4B precursor (MW -35, pi -7.5) was significantly increased post- surgical treatment. To date, two isoforms (MW -31.73-31.94, pi -6.41-6.54) of this protein have been identified by 2-DE. C4B precursor undergoes proteolytic cleavages to produce the mature form of the protein, C4. In its activated form, C4 is a subunit of the C3 and C5 convertases, the enzymatic complexes that activate C3 and C5 of the classical and lectin complement activation pathways. Therefore, production of complement C4 in excess could lead to over activation of the complement pathways and the inflammatory response. Increased expression of C4B precursor in acromegalic patients following surgery may indicate reduced formation of activated C4, and perhaps decreased activation of the complement pathway.

[0029] Definitions

[0030] The term "protein" (also referred to as "polypeptide") is not restricted to the sequences corresponding to the accession numbers provided above, and includes variants and other isoforms thereof. A variant is defined as a naturally occurring variation in the sequence of a polypeptide which has a high degree of homology with the given sequence. A high degree of homology is defined as at least 90%, preferably at least 95% and most preferably at least 99% homology. Protein variants may occur within a single species or between different species. The above proteins are of human origin, but various embodiments encompass use of the corresponding polypeptides from other mammalian species.

[0031] As used herein, the term "isoform" means a molecular form of a given protein, and includes proteins differing at the level of (1) primary structure (such as due to alternate RNA splicing, or polymorphisms); (2) secondary structure (such as due to different co- or post translational modifications); and/or (3) tertiary or quaternary structure (such as due to different sub-unit interactions, homo- or hetero-oligomeric multimerization). For example, differences in mass and/or charge of specific isoforms may be due to posttranslational modifications, including, but not limited to, alkylation, ubiquitination, phosphorylation, and glycosylation.

[0032] "Diagnosing", "prognosing" or "screening" as used herein means providing an indication that a subject may be afflicted with or at risk of developing a disease, particularly a GH disorder such as acromegaly, and includes other terms such as screening for a disease, providing a risk assessment for disease, etc. It will be appreciated that no such technique is perfect and that such diagnosis, prognosis or the like may be confirmed by other procedures such as physical examination, imaging, histological examination of tissue samples, etc. The term "prognosing" as used herein includes providing an assessment or indication of disease in response to a treatment (such as surgery, radiation, pharmaceuticals, and combinations thereof) after initial diagnosis, as an indication of the efficacy of the treatment, risk of the disease returning, severity of disease following treatment, or the like.

[0033] "Panel test" as described herein refers to a group of individual laboratory tests that are related in some way, including, but not limited to, the medical condition they are designed to detect (e.g., acromegaly, or lack thereof), the specimen type (e.g., blood), and the methodology employed by the test (e.g., detection of altered level of a target protein or proteins).

[0034] The term "marker protein", "marker", or "biomarker" includes all biologically relevant forms of the proteins identified, including post-translational modifications resulting in isoforms of a given protein. For example, the marker protein can be present in the body tissue in a glycosylated, phosphorylated, multimeric or precursor form. Marker proteins described herein include any protein listed in Table 1 herein.

[0035] The term "treatment for acromegaly" broadly refers to any therapy intended to 1) reduce the production of GH and/or IGF-1; and or 2) mitigate the negative effects associated with elevated levels of GH on a variety of body tissues including liver, muscle, fat, heart, kidney, brain, and others. Known treatments include, but are not limited to, surgery, such as transsphenoidal surgery to remove GH secreting adenomas of the pituitary gland; medications, such as the somatostatin analogues octreocide and lanreotide, or a GH receptor antagonists termed Pegvisomant; and or radiation therapy.

[0036] The term "differentially expressed" means that the stained protein-bearing spots are present at a higher or lower optical density in the gel from the sample taken for diagnosis (the "diagnostic sample") relative to that from the gel from a control or other comparative sample. In various embodiments, these changes result from a treatment for acromegaly. It follows that the proteins are present in the diagnostic sample at a higher or lower concentration than in the control or other comparative sample. Similarly, "Altered level" or "altered levels" as used with respect to marker proteins herein refers to an increased level (e.g., a one or two fold increase, or more) or a decreased level (e.g., a one or two-fold decrease, or more) in the quantity of one or more marker proteins detectable in or via a biological sample from a post- treatment subject (e.g., subject after transsphenoidal surgery), as compared to a level or levels of one or more marker proteins in the subject prior to treatment or at an earlier stage in treatment. [0037] Some protein "spots" will represent post-translational modifications of the same protein while others may represent heterogeneity due to genetic polymorphisms. For example, 2D gels often reveal a "charge" train representing a difference in isoelectric points of the said protein that may be caused by differential phosphorylation states of the same protein.

[0038] The term "binding partner" includes a substance that recognizes or has affinity for the marker protein. It may or may not itself be labeled.

[0039] The term "antibody" includes polyclonal antiserum, mouse monoclonal antibodies, mouse/human chimeric monoclonal antibodies, humanized monoclonal antibodies, human monoclonal antibodies, and fragments of any of the types of antibodies such as single chain and Fab fragments, and genetically engineered antibodies. The antibodies may be chimeric or of a single species.

[0040] The term "valid body tissue" means any tissue in which it may reasonably be expected that a marker protein would accumulate in relation to a GH disorder. For example, it may be a body fluid such as blood or a blood derivative such as plasma or serum, saliva, or urine.

[0041] Suitable methods for determining an amino acid sequence of the proteins and peptides include, but are not limited to, Edman degradation, (tandem) mass spectrometry and the like (see e.g. Edman, P. Mol. Biol. Biochem. Biophys., (1970), 8: 211-255; U.S. Pat. No.

6,799,121). The amino acid sequence of the proteins and peptides may be compared to amino acid sequences of known proteins. The term " mass spectrometry " as used herein includes various methods such as tandem mass spectrometry, matrix assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry, MALDI-TOF-TOF mass spectrometry, MALDI Quadrupole-time-of-flight (Q-TOF) mass spectrometry, electrospray ionization (ESI)- TOF mass spectrometry , ESI-Q-TOF, ESTTOF-TOF, ESTion trap mass spectrometry , ESI Triple quadrupole mass spectrometry , ESI Fourier Transform mass spectrometry (FTMS), MALDI-FTMS, MALDI-Ion Trap-TOF, and ESI-Ion Trap TOF. These mass spectrometry methods are well known in the art (see e.g. Gary Siuzdak, "Mass Spectrometry for

Biotechnology", Academic Press, NY, (1996)). At its most basic level, mass spectrometry involves ionizing a molecule and then measuring the mass of the resulting ion. Since molecules ionize in a way that is well known, the molecular weight of the molecule can generally be accurately determined from the mass of the ion. Tandem mass spectrometry, for instance, may be used to identify proteins because it can provide information in addition to parent ion molecular weight. Tandem mass spectrometry involves first obtaining a mass spectrum of the ion of interest, then fragmenting that ion and obtaining a mass spectrum of the fragments.

Tandem mass spectrometry thus provides both molecular weight information and a fragmentation pattern that can be used in combination along with the molecular weight information to identify the exact sequence of a peptide or protein (see e.g. Hunt et al. (1986) PNAS USA 83:6233-6237; Shevchenko et al. (1996) PNAS USA 93: 14440-14445; Figeys et al. (1996) Anal. Chem. 68: 1822-1828 and Wilm et al. (1996) Nature 379:466-469.

[0042] "Subjects" as described herein are generally human subjects and includes "patients". The subjects may be male or female and may be of any race or ethnicity, including but not limited to Caucasian, African-American, African, Asian, Hispanic, Indian, etc. The subjects may be of any age, including newborn, neonate, infant, child, adolescent, adult, and geriatric. Subjects may also include animal subjects, particularly mammalian subjects such as dog, cat, horse, mouse, rat, etc., screened for veterinary medicine or pharmaceutical drug development purposes. Subjects include but are not limited to those who may have, possess, or have been previously diagnosed as afflicted with acromegaly.

[0043] "Biological sample" as used herein refers to any material taken from the body of a subject that may carry the target compound or compounds of the tests described herein, including both tissue samples and biological fluids such as blood samples, saliva samples, urine samples, etc. The sample can be taken from any valid body tissue, especially body fluid, of a (human) subject, but preferably blood, plasma or serum. Certain methods disclosed herein involve collecting a biological sample from a subject. The collection of biological samples is performed by standard methods. Typically, once a sample is collected, the biomarkers are detected and measured. The disclosed biomarkers are detected using any suitable technique. Further, molecules that interact with or bind to the disclosed biomarkers, such as antibodies to a biomarker, are detected using known techniques. Many suitable techniques— such as techniques generally known for the detection of proteins, peptides and other analytes and antigens— are known, some of which are described below.

[0044] "Blood sample" as used herein refers to whole blood or any fraction thereof that may contain detectable levels of marker proteins therein (if marker proteins are present in the whole blood sample from which said fraction is obtained), and in particular embodiments refers to a blood sera or blood plasma sample.

[0045] While the following description focuses primarily on acromegaly, it will be appreciated that the embodiments may also be utilized in connection with other disorders in which GH levels are elevated or depressed. For example, if a specific biomarker concentration goes 'down' following successful acromegaly treatment, one of skill in the art would expect the marker to 'go up' following GH administration to normal subjects (e.g., athletes where GH is used as a performance enhancing drug) or following GH administration to GH deficient patients.

[0046] Assay Procedures

[0047] The step of collecting a sample can be carried out either directly or indirectly by any suitable technique. For example, a blood sample from a subject can be carried out by phlebotomy or any other suitable technique, with the blood sample processed further to provide a serum sample or other suitable blood fraction.

[0048] The step of determining the presence of an altered level of a marker protein in the sample, and/or depressed level of a marker protein in the sample, can also be carried out either directly or indirectly in accordance with known techniques, including, but not limited to, mass spectrometry, chromatography, electrophoresis, sedimentation, isoelectric focusing, and antibody assay. See, e.g., U.S. Pat. No. 6,589,748; U.S. Pat. No. 6,027,896.

[0049] In various embodiments, marker proteins may be identified by two-dimensional electrophoresis (2-D electrophoresis). 2D-electrophoresis is a technique comprising denaturing electrophoresis, followed by isoelectric focusing; this generates a two-dimensional gel (2D gel) containing a plurality of separated proteins. For an example of a preferred means of carrying out 2D-electrophoresis to identify marker proteins, see, e.g. WO 98/23950; U.S. Pat. No. 6,064,654 and U.S. Pat. No. 6,278,794. Briefly, spots identified in a 2D gel are characterized by their isoelectric point (pi) and apparent molecular weight (MW) as determined by 2D gel electrophoresis. Altered levels of marker proteins in a first sample or sample set with respect to a second sample or sample set can be determined when 2D gel electrophoresis gives a different signal when applied to the first and second samples or sample sets. Altered levels of marker proteins may be present in first sample or sample sets at increased, elevated, depressed or reduced levels as compared to the second sample or sample sets. By "increased level" it is meant (a) any level of a marker protein when that marker protein is not present in a subject pre- treatment, as well as (b) an elevated level (e.g., a two- or three-fold increase in detected quantity) of marker protein or a particular isoform of a marker protein when that protein or a particular isoform is present in a subject pre-treatment. By "depressed level" it is meant (a) an absence of a particular marker protein or isoform of a particular marker protein when that marker protein is present in a subject pre-treatment, as well as (b) a reduced level (e.g., a two- or three-fold reduction in detected quantity) of a marker protein or isoform of a marker protein when that protein or isoform is present in a subject pre-treatment. In general, the steps of (a) assaying a sample for an elevated level of a marker protein and/or depressed level of a marker protein, and (b) correlating an elevated level of a marker protein and/or a depressed level of a marker protein in the sample with treatment efficacy, can be carried out in accordance with known techniques or variations thereof that will be apparent to persons skilled in the art. See, e.g., U.S. Pat. No. 4,940,658 to Allen et al.

[0050] Signals obtained upon analyzing a biological sample or sample set from pre-treatment subjects having acromegaly relative to signals obtained upon analyzing a biological sample or sample set from the same subjects post-treatment will depend upon the particular analytical protocol and detection technique that is used. Accordingly, the invention contemplates that each laboratory will establish a reference range for each marker protein identifier (e.g., pi and/or MW) in pretreatment acromegaly subjects according to the analytical protocol and detection technique in use, as is conventional in the diagnostic art.

[0051] Kits for monitoring the efficacy of treatments for acromegaly are also provided, and in some embodiments include at least one biochemical material and/or reagent, such as buffers and/or binding partners that are capable of specifically binding with one or more marker proteins from Table 1. These can provide a means for determining binding between the biochemical material and one or more marker proteins, whereby at least one analysis to determine a presence of one or more marker proteins, analyte thereof, or a biochemical material specific thereto, is carried out on a biological sample. Optionally such analysis or analyses may be carried out with the additional use of detection devices for immunoassay, radioimmunoassay, immunoblotting, chromatography, spectrometry, electrophoresis, sedimentation, isoelectric focusing, colorometric, laser, or any combination thereof. Analysis may be carried out on a single sample or multiple samples. In addition, the kit may optionally include instructions for performing the method or assay. Additionally the kit may optionally include depictions or photographs that represent the appearance of positive and negative results. In some embodiments, the components of the kit may be packaged together in a common container. Various embodiments include a kit for monitoring the effect of a treatment for acromegaly in a subject, comprising ligands specific for two or more of transthyretin (P02766; gil55669575), haptoglobin a2 (P00738; gil296653), beta-hemoglobin (Q14484; gil61679768), apoA-I (P02647; gil90108664), and complement C4B precursor (P0C0L5; gill314244). In some embodiments, the ligands are isoform specific antibodies. Such a kit optionally comprises a labeling means and/or a therapeutic agent. Additionally, the kit may include instructional materials for performing various methods presented herein. These instructions may be printed and/or may be supplied, without limitation, as an electronic-readable medium, such as a floppy disc, a CD-ROM, a DVD, a Zip disc, a video cassette, an audiotape, and a flash memory device. Alternatively, instructions may be published on an internet web site or may be distributed to the user as an electronic mail. When a kit is supplied, the different components can be packaged in separate containers. Such packaging of the components separately can permit long term storage without losing the active components' functions.

[0052] Panel Tests

[0053] The marker proteins described herein can be detected individually or in panels with one another or other additional markers for acromegaly. Where used in a panel test, the levels of the various markers are optionally but preferably tested from the same biological sample obtained from the subject (e.g., by detecting the quantities or amounts of various proteins in the same blood sample obtained from a patient). When combined in a panel test, the panel test may include determining an altered level for each of 2, 3, 4, 5, 6, 7 or more different marker proteins (e.g., a panel of some or all proteins set forth in Table 1 below). The combination of multiple marker proteins in a panel test serves to reduce the number of false positives and false negatives should an aberrant value for one particular member of the panel be found.

[0054] Various immunoassays are described in U.S. Pat. No. 7,713,525, incorporated by reference herein. Immunodetection methods may be used for detecting, binding, purifying, removing and quantifying various molecules including the disclosed biomarkers. Further, antibodies and ligands to the disclosed biomarkers are detected. For example, the disclosed biomarkers are employed to detect antibodies having reactivity therewith.

[0055] Immunoassay methods are based on the reaction of an antibody to its corresponding target or analyte and can detect the analyte in a sample depending on the specific assay format. To improve specificity and sensitivity of an assay method based on immuno-reactivity, monoclonal antibodies are often used because of their specific epitope recognition. Polyclonal antibodies have also been successfully used in various immunoassays because of their increased affinity for the target as compared to monoclonal antibodies. Immunoassays have been designed for use with a wide range of biological sample matrices. Immunoassay formats have been designed to provide qualitative, semi-quantitative, and quantitative results.

[0056] Quantitative results are generated through the use of a standard curve created with known concentrations of the specific analyte to be detected. The response or signal from an unknown sample is plotted onto the standard curve, and a quantity or value corresponding to the target in the unknown sample is established.

[0057] Numerous immunoassay formats have been designed. ELISA or EIA can be quantitative for the detection of an analyte. This method relies on attachment of a label to either the analyte or the antibody and the label component includes, either directly or indirectly, an enzyme. ELISA tests may be formatted for direct, indirect, competitive, or sandwich detection of the analyte. Other methods rely on labels such as, for example, radioisotopes (I ) or fluorescence. Additional techniques include, for example, agglutination, nephelometry, turbidimetry, Western blot, immunoprecipitation, immunocytochemistry, immunohistochemistry, flow cytometry, Luminex assay, and others (see ImmunoAssay: A Practical Guide, edited by Brian Law, published by Taylor & Francis, Ltd., 2005 edition).

[0058] Exemplary assay formats include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, fluorescent, chemiluminescence, and fluorescence resonance energy transfer (FRET) or time resolved-FRET (TR-FRET) immunoassays. Examples of procedures for detecting biomarkers include biomarker immunoprecipitation followed by quantitative methods that allow size and peptide level discrimination, such as gel electrophoresis, capillary electrophoresis, planar electrochromatography, and the like.

[0059] Methods of detecting and/or quantifying a detectable label or signal generating material depend on the nature of the label. The products of reactions catalyzed by appropriate enzymes (where the detectable label is an enzyme; see above) can be, without limitation, fluorescent, luminescent, or radioactive or they may absorb visible or ultraviolet light. Examples of detectors suitable for detecting such detectable labels include, without limitation, x-ray film, radioactivity counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, luminometers, and densitometers.

[0060] Any of the methods for detection can be performed in any format that allows for any suitable preparation, processing, and analysis of the reactions. This can be, for example, in multi-well assay plates (e.g., 96 wells or 384 wells) or using any suitable array or microarray. Stock solutions for various agents can be made manually or robotically, and all subsequent pipetting, diluting, mixing, distribution, washing, incubating, sample readout, data collection and analysis can be done robotically using commercially available analysis software, robotics, and detection instrumentation capable of detecting a detectable label.

[0061] One embodiment comprises performing a binding assay for the marker protein. Preferably, an isoform specific binding partner may be used. The binding partner may be labeled. Preferably the assay is an immunoassay, especially between the marker and an antibody that recognizes the protein, or more preferably, the relevant protein isoform, especially a labeled antibody. It can be an antibody raised against part or all of it, for example, a monoclonal antibody or a polyclonal anti-human antiserum of high specificity for the marker protein.

[0062] Thus, the marker proteins described above are useful for the purpose of raising antibodies thereto which can be used to detect the increased or decreased concentration of the marker proteins present in a diagnostic sample. Such antibodies can be raised by any of the methods well known in the immunodiagnostics field.

[0063] The antibodies may be isoform specific, i.e. they recognize specific isoforoms of a given biomarker. Moreover, the antibodies may be anti- to any biologically relevant state of the protein. Thus, for example, they can be raised against the unglycosylated form of a protein which exists in the body in a glycosylated form, against a more mature form of a precursor protein, e.g. minus its signal sequence, or against a peptide carrying a relevant epitope of the marker protein.

[0064] Various immunoassays may be carried out by measuring the extent of the protein/antibody interaction. Any known method of immunoassay may be used. In one embodiment, a sandwich assay may be used. In this method, a first antibody to the marker protein is bound to the solid phase such as a well of a plastics microtitre plate, and incubated with the sample and with a labeled second antibody specific to the protein (or specific protein isoform) to be assayed. Alternatively, an antibody capture assay can be used. Here, the test sample is allowed to bind to a solid phase, and the anti-marker protein antibody is then added and allowed to bind. After washing away unbound material, the amount of antibody bound to the solid phase is determined using a labeled second antibody, anti- to the first.

[0065] The binding partner in the binding assay is preferably a labeled specific binding partner, but not necessarily an antibody. The binding partner will usually be labeled itself, but alternatively it may be detected by a secondary reaction in which a signal is generated, e.g. from another labeled substance.

[0066] It is helpful to use an amplified form of assay, whereby an enhanced "signal" is produced from a relatively low level of protein to be detected. One particular form of amplified immunoassay is enhanced chemiluminescent assay. Conveniently, the antibody is labeled with horseradish peroxidase, which participates in a chemiluminescent reaction with luminol, a peroxide substrate and a compound which enhances the intensity and duration of the emitted light, typically 4-iodophenol or 4-hydroxycinnamic acid.

[0067] EXAMPLES

[0068] The following examples are included to demonstrate embodiments. It should be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention.

[0069] Materials and Methods

[0070] Subjects and Serum Samples

[0071] Eight acromegalic patients (3 female and 5 male) were included in this study and were 26-71 years of age (mean age = 51 yrs). All patients presented with classic symptoms and signs of acromegaly including the presence of a pituitary adenoma visualized by MR imaging. All patients were treated with surgery alone, i.e. none of the patients had received dopamine agonists, somatostatin analogs, or the GH receptor antagonist, Pegvisomant, at any time point, and no patient had received radiation therapy. Serum samples were obtained before and 3-6 months after transsphenoidal surgery. The patients were hospitalized the day before the samples were collected and blood was drawn in the morning after an overnight fast. After incubation for 30-60 min at room temperature, the samples were centrifuged at 3500 g for 10 min at 4 °C. Serum was removed and stored at -20 °C until further analysis.

[0072] All subjects gave a written informed consent before participating in the study, which was approved by The Central Denmark Region Committees on Biomedical Research Ethics (200401184) in adherence to the declaration of Helsinki. The protocol was also approved by the Ohio University Institutional Review Board.

[0073] GH, IGF-1 and total haptoglobin measurements

[0074] Serum GH was measured by a DELFIA assay (PerkinElmer, Tiirku, Finland) and serum IGF-1 levels were determined by an in-house noncompetitive, time-resolved immunofluorometric assay. Both assays have been previously described. Total haptoglobin levels were determined by Cobas c-systems, an immunoassay system (Roche Diagnostics, Mannheim, Germany).

[0075] Sample preparation for proteomic analysis

[0076] Serum samples were shipped frozen on dry ice from Aarhus, Denmark to Athens, OH and stored frozen at -80 °C. In general, all proteomic procedures were performed as described previously. Briefly, serum protein concentrations were determined by the Bradford method. No significant difference in total protein concentration was found between the samples obtained pre- and post-surgery (P>0.05). Albumin depletion of the samples was performed employing a ProteoPrep® Blue Albumin & IgG Depletion Kit (Sigma, St. Lewis, MO) following the manufacturer's instructions. After depletion, 0.3 mg of each sample was diluted in sample buffer containing 7 M urea, 2 M thiourea, 1% w/v SB 3- 10, 3% w/v CHAPS, 0.25% v/v Bio- Lyte 3/10 ampholytes (BioRad Laboratories Inc., Hercules, CA), and 1.5 % v/v protease inhibitor cocktail (Sigma). Disulfide bonds were reduced by adding tributylphosphine. Following reduction, sulfhydryl groups were alkylated with iodoacetamide.

[0077] Two-dimensional gel electrophoresis (2-DE)

[0078] Samples were subjected to 2-DE following procedures previously described (18-21). Serum was transferred to individual wells of an isoelectric focusing (IEF) tray (BioRad, Hercules, CA) with 17 cm IPG strips (pH 3- 10 linear, BioRad, Hercules, CA) and incubated for 2 h at room temperature. IEF was then performed in a PROTEAN IEF cell (BioRad, Hercules, CA), where strips were rehydrated at 50 V for 12 h after which proteins were separated at 1000 V for 60000 V h. Once IEF was complete, the IPG strips were removed and transferred to disposable trays containing 2 ml of equilibration buffer (6 M urea; 2 % SDS; 375 mM Tris-HCl, pH 8.8; 20 % glycerol). The samples were equilibrated with subtle shaking for 45 min. Next, 4.5 cm was cut from each end of the 17 cm IPG strips. The resulting, 8 cm strips (~pH 5-8) were loaded on a 15 % polyacrylamide gel for SDS-PAGE. Proteins were separated in a Mini-PROTEAN 3 cell (Bio-Rad) at 270 V h. Following electrophoresis, the gels were stained using SYPRO Orange (Invitrogen, Carlsbad, CA). Images of stained gels were obtained using a PharosFX Plus Molecular Imager (Bio-Rad, Hercules, CA) with an excitation wavelength of 488 nm and emission detected at 605 nm. Protein spot intensities and matching intensities were performed using the image analysis software PDQuest Advanced v. 8.0 (BioRad, Hercules, CA). Spot intensities were normalized to total image density in each gel, which depended on the total protein content of each sample. Protein spots that were differentially expressed between groups (P < 0.05) were excised from the gels and sent to ProteaBioscience, Inc., Morgantown, WV for identification by mass spectrometry (MS) and tandem-MS (MS/MS) as described below.

[0079] MS analysis

[0080] Protein spots displaying significant (P< 0.05) intensity changes pre- and post- treatment were identified by MS and MS/MS using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) and MALDI-TOF-TOF. Acrylamide gel plugs containing individual spots were dehydrated and then rehydrated with acetonitrile and 50 mM ammonium bicarbonate, respectively. Proteins were then reduced with 250 mM DTT for 60 min at 55 °C, followed by alkylation with 650 mM iodoacetamide for 60 min at room temperature in the dark. Digestion was performed with 500 ng trypsin in 50mM ammonium bicarbonate buffer overnight. Extraction of peptides was performed using 5% formic acid in 50% acetonitrile (dehydration), followed by rehydration with 50 mM ammonium bicarbonate. The procedure was performed three times per sample. The recovered peptides were then lyophilized, reconstituted in 10 mM acetic acid, and re-lyophilized to yield a purified, protein digest extract. For MS and MS/MS analyses, the protein digest solution was loaded onto a CI 8 ProteaTip by aspirating and expelling the sample 5-10 times within the sample vial. The bound sample was washed twice with the 0.1%TFA / 2% acetonitrile solution by aspirating and expelling 20 μΐ of the wash solution 5-10 times. The sample was spotted directly onto a MALDI target that was pre-spotted with 0.6 μΐ MALDI matrix (CHCA) using Ιμΐ of an elution solution (0.1%TFA / 90% acetonitrile). Mass spectra were acquired on an ABI 4800 MALDI TOF/TOF analyzer. MS spectra were acquired in Reflector Positive Ion mode. Peptide masses were acquired for the range from 850-4000 Da. MS spectra were summed from 400 laser shots. Internal calibration was performed using a minimum of three trypsin autolysis peaks. For MS/MS, spectra were acquired until at least 4 peaks in the MSMS spectra achieved a S/N (signal-to-noise ratio) equal to 70.

[0081] Database correlation analysis search parameters

[0082] Protein identification from MS and MS/MS data used the following criteria: Program for MS/MS data processing: ProteinPilot 3.0; Search Engine: Mascot (Matrix Science); Sample Type: gel samples; Digestion Enzyme: Trypsin; Species: Human; Database: NCBInr; Search Engine: Type of Search: Combined MS and MS/MS; Mass Values: monoisotopic; Protein Mass: unrestricted; Peptide Mass Tolerance: + 0.3 - 1 Da; Maximum Missed Cleavages: 1; Variable Modifications: carbamidomethyl (C); Exclusion mass list: 1151.8, 1358.9, 1795.1, 2211.4, 2225.4, 2283.

[0083] Manual protein database searching with MS and MS/MS-generated peak lists

[0084] Protein identities were further verified by using the MS and MS/MS data obtained and the online software named Mascot. Search parameters included the following: MS: database: NCBInr; taxonomy: Homo sapiens; enzyme: trypsin; missed cleavages allowed: 1; fixed modifications: none; protein mass: not specified; peptide tolerance: +1.2 Da; mass values: MH+; monoisotopic/average: monoisotopic. MS/MS: database: NCBInr; taxonomy: Homo sapiens; enzyme: trypsin; missed cleavages allowed: 1; fixed modifications: none; Quantitation: none; peptide tolerance: +1.2 Da; MS/MS tolerance: +0.6 Da; Peptide charge: 1+; monoisotopic/average: monoisotopic; Precursor m/z: not specified; Instrument: MALDI-TOF- TOF. Variable modifications that were included in separate and combined submissions for both MS and MS/MS were Acetyl (K), Carbamidomethyl (C), Deamidated (NQ), Oxidation (M), Phospho (ST), Phospho (Y), Sulfo (S), Sulfo (T), Sulfo (Y). The general criteria used for assessment of protein identity were a minimum significant match of two MS/MS fragment scores (22).

[0085] Western Blot analysis

[0086] Western blot analyses were performed to confirm the identity of haptoglobin, apoA-1 and transthyretin. For 1 -dimension (D) gel electrophoresic immunoblots, 0.05 mg of each sample was loaded onto a SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). For the 2-D western blots, 0.3 mg of each sample was subject to 2-DE and transferred to a PVDF membrane. Membranes were then blocked in 5% non-fat dry milk and probed for 2 h with primary antibody. Antibodies against haptoglobin (mouse monoclonal antibody anti-haptoglobin of human origin, 1 :5000 dilution), apoA-I (mouse monoclonal antibody anti-apoA-I of human origin, 1 :500) and tranthyretin (rabbit polyclonal antibody anti-prealbumin of human origin 1 :500) were obtained from Santa Cruz Biotechnology, Santa Cruz, CA. Protein bands and protein isoforms were identified with horseradish peroxidase-conjugated secondary antibody (1 :5000 dilution) and Pierce ECL western blotting substrate (Thermo scientific, Rockford, IL). The resulting blots were scanned using a Pharos FX Plus Imaging System (BioRad, Hercules, CA) and subjected to image analysis using Quantity One Quantification Program Software (BioRad, Hercules, CA).

[0087] Statistical analysis

[0088] All protein spot intensities identified by 2-DE were analyzed for normality (Shapiro- Wilk test) and variance (homogeneity test for two dependent samples) (23). Protein spots displaying normal distributions and equal variances were compared between the two groups using a two-tailed paired i-test (corresponding P values are reported; see significant spots A, B and D). The non-parametric Wilcoxon signed-ranks test was used to analyze the remaining data (P values are reported; see spots C, E, F and G). GH and IGF- 1 levels between groups were also analyzed using a two-tailed paired i-test. All tests were performed using SPSS v. 14.0. The levels of significance were set at P<0.05.

[0089] Results

[0090] GH and IGF-1 values pre- and post-surgery

[0091] Both serum GH ^g/L) and total IGF- 1 levels ^g/L) decreased significantly (P<0.05) after surgery [GH: 5.29+2.14 (pre) vs. 0.62+0.21 (post); IGF-1 : 637+123.90 vs. 242.25+40.95 (post)] . Results are presented as mean and SEM (Figure 1A-B). The serum GH and IGF-1 levels for each patient pre- and post-treatment are shown in Figure 1. GH levels decreased in seven patients following the surgery, with the exception of patient 2 (Figure 1C). Serum levels of IGF-1 were normalized in five patients following the surgical treatment (Figure ID). Patient 1 presented a decreased in GH [1.49 g/L (pre) vs. 0.53 g/L (post)] but no reduction in IGF-1 levels [211 g/L (pre) vs. 220 g/L (post)]. Patients 6 and 7 showed pronounced reduction in GH serum levels following surgery, whereas the decrease in IGF-1 failed to reach the normal range.

[0092] Serum Proteome changes post-surgery

[0093] The proteomic profiles of all serum samples were evaluated in each subject at pre- and post-treatment time points. Protein profiles on the gels were reproducible and presented similar spot patterns (Figure 2). A total of 150 protein spots were detected in all gels. Protein spot intensities were analyzed for significant changes between pre- and post-surgical samples. Seven protein spots were significantly altered (P<0.05); six decreased and one increased post-surgery (Figure 2).

[0094] Spot intensity changes observed in each individual subject pre- and post-surgery

[0095] Figure 3 (I) shows the average intensity for protein spots A-G before and after the surgery. Protein spots A-F were significantly decreased (P<0.05) following transsphenoidal surgery while protein spot G increased (P<0.05). Spot intensity values pre- and post-surgery showed similar expression patterns (decrease/increase) in all subjects (Figure 3 (II)), suggesting positive correlations between the expression of these specific proteins and the outcome of the surgical procedure.

[0096] Protein identities

[0097] The protein identities of spots A-G (Figure 2) were determined employing MS and MS/MS (Table 1). Among these proteins, two spots located at -15 kDa (A and B) were identified as isoforms of transthyretin (Figure 4 (I)). Both were down-regulated (spot A, P=0.02 and spot B, P=0.01) (Figure 4 (II)) after surgery. Protein Spots C-F also significantly decreased (Figure 4 (II)) in the post-surgery serum samples. Spot C (P=0.04) and D (P=0.02) were identified as haptoglobin a2 (-20 kDa) and beta-hemoglobin (-12 kDa), respectively (Figure 4 (I)). Spots E (P=0.03) and F (P=0.03) located at -10 kDa, were identified as two isoforms of apolipoprotein A-I (apoA-I) (Figure 4 I-II). Protein spot G (-35 kDa) (P=0.04) was identified as complement C4B precursor and was found to be significantly increased after treatment (Figure 4 I-II).

[0098] Total haptoglobin levels and western blot analyses pre- and post-surgery [0099] Total haptoglobin serum levels were quantified (Pre-treatment: 1.08 g/L +0.24; Post- treatment: 1.28 g/L +0.19). No statistically significant differences (P>0.05) were found following transsphenoidal surgery (Figure 5A). In agreement with these results, no differences in total haptoglobin levels were found by western blotting (Figure 5B). In addition, no differences in apoA-I and transthyretin were found by immunoblotting (Figure 5B).

[00100] 2-D western blotting to confirm transthyretin, haptoglobin and apoA-I

[00101] Serum samples were subjected to 2-D western blotting analysis to confirm the

MS identifications of haptoglobin, apoA-I and transthyretin. As shown in figure 6, western results are consistent with the MS results. Transthyretin isoforms were identified in a pi range from -5.2-7.2 at two different MWs (-15 and -35 kDa) (Figure 6 (Til)). Two of these isoforms correspond to protein spots A (MW -15, pi -6.0) and B (MW -15, pi -6.4) identified as transthyretin by MS (Figure 6 Til (1)). Haptoglobin a2 isoforms were identified at MW of -20 kDa and a pi range of -5.7-7.5 (Figure 6 (I- II)). One of the identified isoforms corresponds to spot C (MW -20, pi -6.2) identified as haptoglobin by MS (Figure 6 I II (2)). Finally, apoA-I isoforms were located at a MW of -28 kDa and pi ranging from -5.2-7.8 (Figure 6 Til (3)). Protein spots E and F identified as apoA-I by MS were not detected by western blotting techniques (Figure 6 I (4)).

[00102] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which these embodiments pertain. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of various embodiments, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[00103] The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way. It will be appreciated that there is an implied "about" prior to metrics such as temperatures, concentrations, and times discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings herein. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of "comprise", "comprises", "comprising", "contain", "contains", "containing", "include", "includes", and "including" are not intended to be limiting. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

OTHER EMBODIMENTS

[00104] It is to be understood that while embodiments have been described in

conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Table 1: Mass spectrometry for spots that changed significantly after treatment

Spot Gel Identity match Accession % of MS Max Matched MS/MS Max Matched pI/MW No. change³ Score^b sequence fragments Score^c sequence fragments coverage coverage

( ) ( )

11 /52

87 67 6 ?

11 6,5

58

6/31

Hemoglobin

a) Percentage (%) of change was calculated dividing the post-treatment mean intensities over the pre-treatment mean values. b) A minimum MS score >64 was considered significant.

c) A minimum of two significant MS/MS peptide fragments was considered to assign an ID for a spot.

d) Spot G was identified as complement C4B precursor based on the protein score obtained by ProteinPilot 3.0.

Claims

1. A method of monitoring the efficacy of a treatment for a GH disorder in a subject, comprising:

(a) detecting one or more markers in a biological sample of the subject, said markers comprise a protein isoform selected from the group consisting of transthyretin (Swiss-Prot Acc. No. P02766), haptoglobin a2 (Swiss-Prot Acc. No. P00738), beta-hemoglobin (Swiss-Prot Acc. No. Q14484), apoA-I (Swiss-Prot Acc. No. P02647), and complement C4B precursor (Swiss-Prot Acc. No. P0C0L5); and

(b) determining an altered levels of said marker(s), said altered levels indicating efficacy of the treatment.

2. The method of claim 1, wherein said one or more markers are included in a diagnostic panel including at least two of transthyretin (Swiss-Prot Acc. No. P02766), haptoglobin a2 (Swiss-Prot Acc. No. P00738), beta-hemoglobin (Swiss-Prot Acc. No. Q14484), apoA-I (Swiss-Prot Acc. No. P02647), and complement C4B precursor (Swiss-Prot Acc. No.

P0C0L5).

3. The method of claim 1, wherein said altered levels are compared to a control sample taken from the same subject prior to said treatment or at an earlier stage in the treatment.

4. The method of claim 1, wherein the detecting step is carried out by immunoassay, chromatography, spectrometry, electrophoresis, sedimentation, isoelectric focusing, or any combination thereof.

5. The method according to any of claims 1-4, wherein the detecting step further comprises the steps of subjecting the biological sample to two-dimensional gel

electrophoresis to yield a stained gel and an increased or decreased concentration of the protein isoform is detected by an increased or decreased intensity of a protein-containing spot on the stained gel, compared with a corresponding control gel.

6. The method of claim 5, wherein an altered level of spot A, B, C, D, E, F or G in FIG. 2 is detected.

7. The method of claim 6, wherein the protein isoform of spot A has a MW of about 15 and a pi of about 6.0; the protein isoform of spot B has a MW of about 15 and a pi of about 6.4; the protein isoform of spot C has a MW of about 20 and a pi of about 6.2; the protein isoform of spot D has a MW of about 12 and a pi of about 7.8; the protein isoform of spot E has a MW of about 10 and a pi of about 5.0; the protein isoform of spot F has a MW of about 10 and a pi of about 5.4; and the protein isoform of spot G has a MW of about 35 and a pi of about 7.5.

8. The method of claim 6, wherein the decreased intensity of spot A, B, C, D, E, or F or the increased intensity of spot G in FIG. 2 is detected.

9. The method according to any of claims 1-8, wherein the sample is a blood sample.

10. The method according to any of claims 1- 9, wherein:

the GH disorder is acromegaly; and

the method further comprises the step of identifying a subject suffering from or at risk for acromegaly.

11. A method of monitoring the effect of a treatment for acromegaly in a patient, which comprises detecting a change in concentration of at least one protein in a sample of a valid body tissue taken from the subject at a stage in said treatment, compared with the

concentration of the protein in a sample of a valid body tissue taken from the subject prior to said treatment or at an earlier stage in the treatment, the protein selected from the group consisting of: transthyretin (Swiss-Prot Acc. No. P02766), haptoglobin a2 (Swiss-Prot Acc. No. P00738), beta-hemoglobin (Swiss-Prot Acc. No. Q14484), apoA-I (Swiss-Prot Acc. No. P02647), and complement C4B precursor (Swiss-Prot Acc. No. P0C0L5).

12. The method of claim 11, wherein the change in protein concentration is a decrease and the protein is selected from the group consisting of: transthyretin (Swiss-Prot Acc. No. P02766), haptoglobin a2 (Swiss-Prot Acc. No. P00738), beta-hemoglobin (Swiss-Prot Acc. No. Q14484), and apoA-I (Swiss-Prot Acc. No. P02647).

13. The method of claim 11, wherein the valid body tissue is blood or a blood product such as serum or plasma.

14. The method according to any of claims 11-13, wherein the detecting step further comprises the steps of subjecting the biological sample to two-dimensional gel

electrophoresis to yield a stained gel and an increased or decreased concentration of the protein isoform is detected by an increased or decreased intensity of a protein-containing spot on the stained gel, compared with a corresponding control gel.

15. The method according to claim 14, wherein an altered level of spot A, B, C, D, E, F, or G in FIG. 2 is detected.

16. The method according to claim 15, wherein the decreased intensity of spot A, B, C, D, E, or F or the increased intensity of spot G in FIG. 2 is detected.

17. The method of claim 11, wherein the treatment comprises a surgery.

18. The method of claim 11, wherein the treatment comprises a radiation therapy.

19. The method of claim 11, wherein the treatment comprises a pharmaceutical agent.

20. A method for predicting the efficacy of a surgical treatment for acromegaly in a subject with acromegaly, comprising:

administering a surgical treatment to the subject with acromegaly; acquiring a diagnostic sample from the subject;

detecting a decreased concentration of a protein in the diagnostic sample, compared to a pre-treatment sample, the protein selected from the group consisting of transthyretin (Swiss-Prot Acc. No. P02766), haptoglobin a2 (Swiss- Prot Acc. No. P00738), beta-hemoglobin (Swiss-Prot Acc. No. Q14484), and apoA-I (Swiss-Prot Acc. No. P02647), the decreased concentration in one or more of the proteins indicating efficacy of the surgical treatment;

or detecting an increased concentration of a complement C4B precursor protein (Swiss-Prot Acc. No. P0C0L5) in the diagnostic sample, compared to a pre-treatment sample, an increased concentration of the complement C4B precursor protein indicating efficacy of the surgical treatment.

21. The method according to claim 20, wherein the diagnostic sample is subjected to two- dimensional gel electrophoresis to yield a stained gel and the increased or decreased concentration of the protein is detected by an increased or decreased intensity of a protein- containing spot on the stained gel, compared with a corresponding control gel.

22. The method according to claim 21, wherein the decreased intensity of spot A, B, C, D, E, or F or the increased intensity of spot G in FIGS. 3 is detected.

23. A kit for monitoring the effect of a treatment for acromegaly in a subject, comprising ligands specific for two or more of transthyretin (Swiss-Prot Acc. No. P02766), haptoglobin a2 (Swiss-Prot Acc. No. P00738), beta-hemoglobin (Swiss-Prot Acc. No. Q14484), apoA-I (Swiss-Prot Acc. No. P02647), and complement C4B precursor (Swiss-Prot Acc. No.

P0C0L5).

24. The kit of claim 23, wherein the ligands are antibodies.

25. The kit of claim 24, wherein the antibodies are isoform specific monoclonal antibodies.