WO2014201220A1 - Procédés de détection du cancer de la prostate - Google Patents

Procédés de détection du cancer de la prostate Download PDF

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WO2014201220A1
WO2014201220A1 PCT/US2014/042074 US2014042074W WO2014201220A1 WO 2014201220 A1 WO2014201220 A1 WO 2014201220A1 US 2014042074 W US2014042074 W US 2014042074W WO 2014201220 A1 WO2014201220 A1 WO 2014201220A1
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arsb
subject
tissue
prostate
recurrence
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Joanne K. Tobacman
Sumit BATTACHARYYA
Leonid FEFERMAN
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University of Illinois at Urbana Champaign
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/06—Sulfuric ester hydrolases (3.1.6)
    • C12Y301/06001—Arylsulfatase (3.1.6.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57555—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)

Definitions

  • Cancer staging is used to assess the severity of a cancer, to predict the likelihood of recurrence, and to select treatment.
  • prostate biopsy results including Gleason scores
  • PSA prostate-specific antigen
  • the predictive value of a diagnostic test is a function of its sensitivity and specificity.
  • PSA has been widely used as a biomarker of prostate cancer
  • the use of PSA to screen for or stage prostate cancer remains controversial because PSA levels are of questionable predictive value.
  • the present invention satisfies this demand.
  • arylsulfatase B As a biomarker of prostatic malignancy was investigated. Measurements of the intensity of ARSB immunostaining by digitized image analysis (H-scores) were calculated for prostate cancers in two small tissue microarrays (TMA) and the associations of H-scores with recurrence vs. non- recurrence and Gleason score were determined. In addition, ARSB enzyme activity, chondroitin-4-sulfate (C4S), and versican were compared between normal and malignant regions from radical prostatectomies performed for prostate cancer. The study data that follow show the usefulness of ARSB as a biomarker and possible tumor suppressor in prostate cancer.
  • H-scores digitized image analysis
  • C4S chondroitin-4-sulfate
  • versican were compared between normal and malignant regions from radical prostatectomies performed for prostate cancer.
  • this invention is a method of predicting the severity or likelihood of recurrence of prostate cancer in a subject comprising i) measuring the amount of ARSB in a sample of prostate tissue from the subject; and ii) comparing the amount of ARSB in the tissue sample to the amount of ARSB in a control sample, wherein a decrease in the amount of ARSB in the tissue sample relative to the amount of ARSB in the control is indicative of the severity or likelihood of recurrence of prostate cancer in the subject.
  • this invention is a method of predicting the severity or recurrence of prostate cancer in a subject comprising i) measuring the ARSB activity in a sample of prostate tissue from the subject; and ii) comparing the ARSB activity in the tissue sample to the ARSB activity in a control sample, wherein a decrease in the ARSB activity in the tissue sample relative to the ARSB activity in the control is indicative of the severity or recurrence of prostate cancer in the subject.
  • the amount of ARSB in the prostate tissue samples is determined by ARSB immunostaining.
  • the amount of ARSB in the prostate tissue is determined spectrophotometrically.
  • H-scores are calculated from the prevalence and intensity of the immunostaining.
  • the H-scores are used to predict the severity or likelihood of recurrence of prostate cancer in the subject.
  • the prostate tissue from the subject is malignant prostate tissue.
  • the prostate tissue from the subject comprises a mixture of normal and malignant prostate tissue.
  • control comprises samples of normal prostate tissues obtained from existing tissue sources, samples of normal prostate tissues obtained from the subject and/or samples of prostate tissue obtained from the subject at an earlier timepoint.
  • control obtained as described above comprises a mixture of normal and malignant prostate tissues.
  • the method further comprises measuring the subject's
  • PSA values wherein a decrease in the amount of ARSB in the subject's prostate tissues and/or the ARSB activity in the subject's prostate tissues relative to the control amount in combination with PSA values is indicative of the severity or likelihood of recurrence of prostate cancer in the subject.
  • Fig. 1A is a graph showing mean ARSB H-scores for stroma, epithelium, and combined stroma and epithelium for 22 pairs (recurrent vs. non-recurrent) of prostate cancer cases.
  • Fig. 1 B. is a scattergram showing the ARSB H-scores in the 22 pairs of prostate cancer cases for epithelium and stroma combined.
  • NDRI National Disease Research Interchange
  • Fig. 2B-E are images of representative prostate cancer cores immunostained for ARSB, with Gleason scores of 6 (Fig. 2B), 7 (Fig. 2C), 8 (Fig. 2D) or 9 (Fig. 2E).
  • Fig. 2F and Fig. 2G. are images of representative TMA cores stained with ARSB polyclonal rabbit antibody or with control IgG.
  • Fig. 2H is a plot of ARSB H-scores vs. Gleason scores (7-9) for the NDRI tissue cores.
  • Fig. 3A is a Western blot using normal (N) and malignant (C) prostate tissue.
  • Fig. 3B is a densitometric scan of the Western blot of Fig. 3B.
  • Fig. 3C is a graph showing ARSB activity in normal and malignant prostate tissue.
  • Fig. 3D is a graph of total sulfated glycosaminoglycans (GAGs) and the chondroitin-4-sulfate (C4S) in normal and malignant prostate tissue.
  • GAGs total sulfated glycosaminoglycans
  • C4S chondroitin-4-sulfate
  • Fig. 3E and Fig. 3F are images of normal and malignant prostate tissue immunostained using C4S antibody.
  • Fig. 3G is a negative control.
  • Fig. 4A is a graph of versican protein levels in normal and malignant prostate tissue.
  • Fig. 4B is a graph showing levels of chondroitin-4-sulfate immunoprecipitated with versican in normal and malignant prostate tissue.
  • Fig. 5A shows EGFR that co-immunoprecipitated with versican as a function of total EGFR (Fig. 5B) in normal and malignant prostate tissue.
  • Arylsulfatase B (ARSB) or N-acetylgalactosamine-4-sulfatase has been described as a lysosomal enzyme that catalyzes the removal of the 4-sulfate group of N-acetylgalactosamine-4-sulfate at the non-reducing end of C4S, and dermatan sulfate (DS), thereby regulating degradation of C4S and DS.
  • Extra-lysosomal localization of ARSB in epithelial and endothelial membranes has been demonstrated in human cells.
  • Reduced ARSB activity has been found in malignant mammary and colonic epithelial tissues and in metastatic colonic epithelial cells, and the intensity and localization of ARSB immunostaining is reduced in higher grade colonic adenocarcinomas.
  • ARSB is reduced in malignant prostate tissue, and that ARSB may serve as a marker for prostate cancer.
  • TMA cancer tissue microarray
  • NDRI National Disease Research Interchange
  • CPCTR Cooperative Prostate Cancer Tissue Resource
  • Tissue microarray slides were hydrated using xylene and an alcohol gradient, and rinsed in distilled water. Antigen unmasking was performed with a 10X concentrated retrieval solution by Dako (DakoCytomation, Carpenteria, CA), according to the manufacturer's instructions, then slides were rinsed in phosphate- buffered saline (PBS) for 5 minutes.
  • PBS phosphate- buffered saline
  • Endogenous peroxidase activity was blocked by H 2 O 2 blocking reagent for 10 minutes at room temperature, then the TMA slides were treated with a protein blocking solution for 10 minutes at room temperature, rinsed and incubated with arylsulfatase B polyclonal rabbit antiserum (Open Biosystems, ThermoFisher Scientific, Huntsville, AL; 1 :100) or negative IgG control for 30 minutes at room temperature. Slides were rinsed and then treated with EnVision Plus labeled polymer (DakoCytomation) for 30 minutes at room temperature.
  • DAB Plus (DakoCytomation) was used for 10 minutes to detect ARSB, and slides were rinsed in distilled water, counterstained with hematoxylin, dehydrated through an alcohol gradient and mounted with Permount.
  • the TMA slides were digitally scanned at 20x magnification on an Aperio ScanScope® CS (Aperio Technologies, Inc., Vista, CA) using the Aperio ImageScope program (v10.0.35.1800) and loaded into Spectrum version 11.1.
  • Other prostate cancer and normal tissue sections from frozen tissue were immunostained with ARSB polyclonal antibody. Negative IgG controls were also prepared and imaged.
  • the TMA slides were digitally scanned at 20x magnification on an Aperio ScanScope® CS (Aperio Technologies, Inc., Vista, CA) using the Aperio ImageScope program (v10.0.35.1800) and loaded into Spectrum version 11.1.
  • the TMA Lab® software module was used to segment the TMAs into individual cores, while the Genie® module was used to map distinct epithelial and stromal regions within each core.
  • Genie® is a machine learning program that classifies each pixel in an image according to a set of hand-drawn, pre-classified training images provided by a skilled human operator. For this study, three classes were created: epithelial, stromal, and no-tissue, using 16, 7 and 1 training images, respectively.
  • the resulting classifier algorithm which was determined to be highly accurate in classifying pixels within the training set of images, was then applied to the entire TMAs. Once epithelial and stromal regions were mapped, it was possible to score staining solely within epithelial or stromal compartments or in combination.
  • the Positive Pixel Count® (Aperio, Inc.) algorithm was used within the epithelial and stromal compartments to measure brown chromogen staining in each relevant pixel at four ordinal intensity levels, from 0 to 3.
  • the H-score an index that combines stain prevalence and intensity, was determined based on the proportion of weakly, moderately and strongly stained pixels in each core using the formula: (% weak x 1 + % moderate x 2 + % strong x 3) / 100.
  • the H-scores were calculated independently for the NDRI and CPCTR cores. Mean H-score ⁇ standard deviation (S.D.) for Gleason scores 6-9 in the NDRI TMA was calculated. The H-scores for the recurrent vs. non-recurrent member of the paired samples in the CPCTR were compared. When multiple cores from the same surgery were present on an array, the H-scores for the cores were averaged, and the average value used in subsequent analysis. Each TMA core was reviewed visually before scoring to exclude artifacts or missing tissue, and again after scoring. No gross discrepancies with the automated scoring were identified.
  • Tissue homogenates were prepared from the normal and malignant foci isolated from the prostatectomies performed at UIC.
  • Arylsulfatase B (ARSB) activity was determined using a fluorometric assay and the exogenous substrate 4- methylumbeliiferyl sulfate, as previously detailed (Bhattacharyya et al., J Steroid Biochem Mol Biol 2007; 103: 20-34, which is incorporated by reference in its entirety). Briefly, 20 ⁇ of tissue homogenate and 80 pi of assay buffer (0.05 M Na acetate buffer, pH 5.6) were combined with 100 ⁇ of substrate (5mM 4-MUS in assay buffer) in wells of a microplate.
  • assay buffer 0.05 M Na acetate buffer, pH 5.6
  • ARSB activity was expressed as nmol/mg protein/hour, based on a standard curve for ARSB activity prepared with known quantities of 4-methylumbilleferyl at pH 5.6. Protein content of the tissue homogenate was determined by total protein assay kit (Pierce, Thermo Fisher Scientific, Inc., Rockford, IL).
  • Western blot for ARSB was performed using paired normal and malignant prostate tissue samples from three of the UIC cases. Tissue lysates were prepared from prostate tissue with cell lysis buffer (Cell Signaling Technology, Inc., Danvers, MA) and protease and phosphatase inhibitors (HaltTM Protease and Phosphatase Inhibitor Cocktail, Thermo Scientific, Pittsburgh, PA). Western blot of ARSB was performed on 10% SDS gel with ARSB antibody, as above, and ⁇ -actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). An ARSB inhibitory peptide with the sequence used to generate the anti-ARSB antibody, was added to three of the wells to verify the specificity of the ARSB band.
  • the sequence of the inhibitory peptide used in the Western blot is RLQFYHKHSVPVYFPAQDPR (SEQ ID NO:1) (NP_15848.1 ; AA: 501-520). Immunoreactive bands were visualized using enhanced chemiluminescence (Amersham, GE Healthcare, Piscataway, NJ, USA). Density of the ARSB was compared to ⁇ -actin in the malignant and normal samples.
  • ARSB measurements were performed following a standard protocol with the synthetic substrate, p-nitrocatechol sulfate.
  • the reaction utilized the substrate 0.1 M p-nitrocatechol sulfate (NCS) in H 2 0 and the buffer 1.0 M Na acetate at pH 6.0 with
  • the extent of the reaction was measured by optical density (OD) readings at 515 nm in the Beckman DU65 spectrophotometer. Enzymatic activity was calculated using the constant 0.0112 to solve for activity expressed as nmol/min/mg protein. Protein content of the cell lysate was measured using BCATM Protein Assay Kit (Pierce).
  • GAG Total sulfated glycosaminoglycan (GAG) content in normal and malignant prostate tissues was measured using the substrate 1 ,9-dimethylmethylene blue (BlyscanTM, Biocolor Ltd., Newtownabbey, Northern Ireland), which detects chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, and heparin, but does not detect unsulfated glycosaminoglycans or disaccharides.
  • BlyscanTM substrate 1 ,9-dimethylmethylene blue
  • the substrate 1 ,9-dimethylmethylene blue combines with the sulfate groups of the sulfated GAG and detects the sulfated polysaccharide component of proteoglycans and the protein-free sulfated GAG chains.
  • Tissue lysates were prepared using RIPA buffer (50 mmol/L Tris-HCI containing 0.15 mol/L NaCI, 1% Nonidet P40, 0.5% deoxycholic acid and 0.1 % SDS, pH 7.4). Absorbance maximum of 1 ,9-dimethylmethylene blue was detected at 656 nm (FLUOstar), and sulfated GAG concentration expressed as pg/mg of protein of tissue lysate.
  • Tissue lysates were prepared using RIPA buffer, as above.
  • Antibody specific to native chondroitin-4-sulfate (C4S; 4D1 , Abnova, Littleton, CO) was previously tested by the recovery of pure C4S following immunoprecipitation with the C4S antibody (1 pg) and shown to be 93.3 ⁇ 2.7%.
  • Cross-reactivity of the antibody with CS-E or C6S was excluded by similar tests.
  • the C4S antibody (1 pg/mg of cell lysate protein) was added to the prostate cell lysates in tubes, and tubes were rotated overnight in a shaker at 4°C.
  • Human versican was measured by a competitive ELISA (My BioSource, San Diego, CA), in which color development was inversely proportional to the versican content in the test samples.
  • Standards ranging from 1 to 25 ng/ml (Mg/L), tissue samples, and versican-horseradish peroxidase conjugate were added to wells pre- coated with versican antibody, incubated for 1 hour at 37°C, and washed three times. Color was developed by adding hydrogen peroxide/ tetramethylbenzidine (TMB) substrate. The reaction was stopped by 2N sulfuric acid, and the color was read at 450 nm in a plate reader (BMG). The concentration of versican in the samples was extrapolated from the standard curve and expressed per mg of total tissue protein, measured by protein assay (Pierce).
  • Total human epidermal growth factor receptor (phosphorylated and unphosphorylated EGFR and ErbB1 ) was measured in the tissue extract using a standardized ELISA (R&D, Minneapolis, MN).
  • Total EGFR in the samples was captured in the wells of microtiter plates that were pre-coated with specific capture antibody.
  • the immobilized total EGFR was detected by a biotinylated second EGFR, and Streptavidin-hydrogen peroxidase (HRP) was added.
  • the bound enzyme activity was determined by chromogenic substrate [(hydrogen peroxide/tetramethylbenzidine (TMB)], and color development due to HRP activity was stopped by 2N sulfuric acid. Intensity of color was measured at 450 nm in a plate reader (BMG), and the values of the samples were extrapolated from a standard curve and normalized using the total tissue protein concentration as measured by protein assay (Pierce).
  • Versican was immunoprecipitated from tissue lysates using versican antibody (V0 isoform; SCBT, Santa Cruz, CA) covalently bound to Dynabeads (Life Technologies, Carlsbad, CA). Total versican concentration in the immunoprecipitate was determined by competitive ELISA as described above. Prostate cancer samples were diluted by 50% in diluent to bring versican to approximately the same concentration as in the normal tissue immunoprecipitates. Blyscan assay for C4S was conducted as described above to detect the C4S that co-immunoprecipitated with versican. Immunohistochemistry of chondroitin-4-sulfate
  • Tissue sections were prepared from the frozen normal and malignant prostate tissues and immunostained with chondroitin-4-sulfate (C4S) mouse monoclonal antibody (4D1 clone, SCBT; 1 :100). Sections were incubated with primary antibody or IgG negative control overnight at 4°C, then washed, then incubated with secondary antibody which was conjugated with HRP for 1 h at room temperature. Color was developed following wash with 3,3-diaminobenzidine and counterstained with hematoxylin. Digitized images were obtained with QCapture software (Qlmaging, Surrey, BC, Canada) at 20X magnification. Background color was modified with GIMP Portable software (Portable Apps, New York, NY).
  • C4S chondroitin-4-sulfate
  • Results are expressed as mean ⁇ standard deviation. Statistical significance of differences in H-scores between paired samples that varied by recurrence vs. non-recurrence and association between H-scores and Gleason scores was determined using Instat (GraphPad Software, San Diego, CA) by either paired or unpaired t-tests, two-tailed, or by one-way analysis of variance, followed by the Tukey-Kramer post-test to correct for multiple comparisons. Paired t-tests were performed with 6 pair of normal and malignant biological samples using averages of technical duplicates of each measurement. P-value of less than 0.05 was considered statistically significant. One asterisk represents p ⁇ 0.05, ** represents p ⁇ 0.01 , *** p ⁇ 0.001 , and ** ** p ⁇ 0.0001.
  • Prostate cancer cases in the CPCTR array were paired for age ⁇ 5 years, race, treatment intervention, Gleason scores (in same sequence), and pathological TNM stage, and were differentiated only by biochemical (elevated PSA) recurrence vs. non-recurrence after 4 or more years of followup.
  • Arylsulfatase B (ARSB; N- acetylgalactosamine-4-sulfatase) immunostaining of the tissue microarray containing 22 pairs of cores from prostatectomies was performed.
  • ARSB H-scores for epithelium, stroma, and combined epithelium and stroma were determined by digitized image analysis for each case, and were compared between the recurrent and non-recurrent members of the pairs. 82% (18/22) of the pairs had higher H- scores for the non-recurrence than for recurrence using the combined stroma and epithelium ARSB H-score (Table 1). In contrast, initial PSA values were lower in only 65% (13/20) of the recurrences, indicating that PSA is less effective as a predictor of recurrence than the ARSB H-score. The combination of higher ARSB H-score and lower PSA value predicted 95% (21/22) of the recurrences.
  • ARSB H-scores were calculated for stroma, epithelium, and combined stroma and epithelium for the 22 pairs of prostate cancer cases that differed by recurrence vs. non-recurrence at 4 or more years of follow-up. Recurrences had lower mean ARSB H-scores for stroma (0.10 ⁇ 0.08 vs. 0.19 ⁇ 0.09), epithelium (0.48 ⁇ 0.32 vs. 0.86 ⁇ 0.24) and combined stroma and epithelium (0.28 ⁇ 0.26 vs.
  • Fig. 1 B shows the ARSB H-scores in the 22 pairs of prostate cancer cases for epithelium and stroma combined. H-scores of less than 0.25 accurately predicted recurrence, and H-scores of greater than 0.70 were associated with non-recurrence in 8 of 9 cases.
  • ARSB H-scores less than 0.25 for combined predicted recurrence with 100% specificity, and H-scores greater than 0.70 for combined were highly predictive of non-recurrence (Fig. 1 B). Overlap between the scores for recurrence and non-recurrence was evident between 0.25 and 0.70, and over 50% (23/44) of the cases were in this range.
  • Mean ARSB H-scores for cores on the NDRI array were calculated and associated with the corresponding Gleason scores. 11 cores were designated Gleason 6, 6 were Gleason 7, 8 were Gleason 8, and 5 were Gleason 9.
  • the ARSB positive epithelial cell membrane becomes increasingly less continuous and more punctate with increasing Gleason score. Overall intensity of stromal and epithelial staining declined with increasing Gleason score.
  • Representative TMA cores demonstrate positive staining for ARSB (Fig. 2F) and negative staining with IgG control (Fig. 2G).
  • Linear regression analysis demonstrated inverse correlations for ARSB H- scores and Gleason scores (7-9) for combined epithelium and stroma, epithelium, and stroma (Fig. 2H).
  • the r-values were -0.99, -0.97, and -0.98, respectively.
  • PSA at diagnosis ranged from 4.3 to 25.4 ng/ml (pg/L)
  • Gleason scores ranged from 6-9.
  • the ARSB activity in the malignant prostate tissue was ⁇ 50% of the value in the normal tissue.
  • Mean ARSB activity in the normal tissue was 39.3 ⁇ 13.4 nmol/mg protein/h, compared to 76.1 ⁇ 7.1 ng/mg protein/h in the malignant tissue (p ⁇ 0.0001 , paired t-test, two-tailed) (Fig. 3C).
  • C4S chondroitin 4-sulfate
  • GAGs glycosaminoglycans
  • Immunohistochemistry of C4S also demonstrates less intense staining of C4S staining in the normal (Fig. 3E), compared to the malignant prostate tissue (Fig. 3F), consistent with the decline in ARSB in the malignant tissue and the resultant increase in C4S. IgG control staining is negative (Fig. 3G).
  • Versican an extracellular matrix proteoglycan with chondroitin sulfate attachments, has previously been considered as a biomarker of prostate cancer. Measurements of versican showed significant increases in versican in the malignant prostate tissues, compared to levels in the normal tissues. The mean value of the increase between the malignant and the normal paired tissues was 119.4 ng/mg protein, an increase of more than 76% over the baseline (p ⁇ 0.0001 , paired t-test, two-tailed) (Fig. 4A).
  • the G2 domain of versican has sites where chondroitin sulfate attaches, and versican isoforms with differences in these attachment sites have been associated with changes in cell proliferation and apoptosis.
  • C4S that co-immunoprecipitated with versican was measured.
  • C4S increased to 2.6 times the level in the normal prostate tissue (Fig. 4B). This increase is consistent with the marked decline in ARSB activity and the overall increase in C4S content in the malignant tissue.
  • ARSB immunostaining determined by digitized analysis, was inversely associated with Gleason scores for epithelial and stromal compartments separately and in combination. Also, when ARSB activity was determined in normal and malignant regions of prostatectomies, ARSB activity was significantly less in the malignant compared to normal tissue. In association with reduced ARSB activity, total sulfated glycosaminoglycans and chondroitin-4-sulfate content were increased in the malignant prostatic tissue, and the chondroitin-4- sulfate containing matrix proteoglycan versican was also increased.
  • Versican was previously reported as a biomarker of prostate malignancy, and increases in versican and in chondroitin sulfate have been identified as predictors of disease progression. This report suggests that the decline in ARSB activity and the associated increase in C4S may impact versican-associated processes in the stroma and on the stromal-epithelial interactions.
  • stromal versican can interact with epithelial cell surface receptors and can modulate signaling pathways, including the EGF-EGFR pathway, since two epidermal growth factor (EGF)-like motifs are located at the C-terminus of the G3 domain of versican.
  • EGF epidermal growth factor
  • ARSB and PSA may be more informative than either test alone. Analysis of larger databases with outcome data will provide confirmation that ARSB activity and/or ARSB immunohistochemical scores can help to predict recurrence and severity of disease. Using the teachings disclosed herein, it is well within the ability of one of ordinary skill in the art to identify specific cutoffs for ARSB H-scores or for ARSB activity for recurrence or non- recurrence. Standardization of H-scores may be difficult, since variation in the range of H-scores was present in the two small arrays analyzed in this report. Potentially, microgram quantities of tumor tissue from biopsy samples can be studied to determine ARSB activity and to correlate activity with outcome data. Because ARSB treatment has been used in the treatment of people with mucopolysaccharidosis VI, a condition caused by a deficiency of ARSB, it is envisioned that ARSB may be administered in therapeutic amounts to treat prostate cancer.

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  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
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  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Analytical Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
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Abstract

L'invention concerne des procédés de prévision de la gravité ou de la probabilité de récurrence du cancer de la prostate chez un sujet atteint d'un cancer de la prostate par mesure de la quantité d'activité de la N-acétylgalactosamine-4-sulfatase dans un tissu de cancer de la prostate et la comparaison de la quantité ou de l'activité à celle d'un échantillon témoin, une quantité ou une activité réduite indiquant la gravité ou la probabilité de récurrence du cancer de la prostate chez le sujet.
PCT/US2014/042074 2013-06-12 2014-06-12 Procédés de détection du cancer de la prostate Ceased WO2014201220A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060078959A1 (en) * 2004-03-10 2006-04-13 Massachusetts Institute Of Technology Recombinant chondroitinase ABC I and uses thereof
US20120189605A1 (en) * 2010-07-22 2012-07-26 Biomarin Pharmaceutical Inc. Manufacture of active highly phosphorylated human n-acetylgalactosamine-6-sulfatase and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060078959A1 (en) * 2004-03-10 2006-04-13 Massachusetts Institute Of Technology Recombinant chondroitinase ABC I and uses thereof
US20120189605A1 (en) * 2010-07-22 2012-07-26 Biomarin Pharmaceutical Inc. Manufacture of active highly phosphorylated human n-acetylgalactosamine-6-sulfatase and uses thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FEFERMAN, L ET AL.: "Reduced Arylsulfatase B Enzyme Activity And Immunostaining In Malignant Prostate Tissue.", CARCINOGENESIS AND CHEMOPREVENTION, 6 March 2012 (2012-03-06), CHICAGO, IL *
RICCIARDELLI, C ET AL.: "Elevated Stromal Chondroitin Sulfate Glycosaminoglycan Predicts Progression In Early-Stage Prostate Cancer.", CLIN. CANCER RES., vol. 3, no. 6, June 1997 (1997-06-01), pages 983 - 992 *

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