Patent Application: US-201414899352-A

Abstract:
the present invention relates to the field of cancer . more specifically , the present invention provides methods and compositions useful for assessing prostate cancer in a patient . in a specific embodiment , a method for predicting metastasis in a prostate cancer patient comprises the steps of genotyping the aspn d repeat domain length in both alleles of the patient using a polymerase chain reaction ; predicting metastasis in the patient if the aspnd repeat domain length is 13 in one allele and 14 in the other allele or have at least one allele with 14 d repeats as compared to other common allelic genotypes ; and predicting no metastasis in the patient if the aspn d repeat domain length is 13 in both alleles as compared to other allelic genotypes .

Description:
it is understood that the present invention is not limited to the particular methods and components , etc ., described herein , as these may vary . it is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only , and is not intended to limit the scope of the present invention . it must be noted that as used herein and in the appended claims , the singular forms “ a ,” “ an ,” and “ the ” include the plural reference unless the context clearly dictates otherwise . thus , for example , a reference to a “ protein ” is a reference to one or more proteins , and includes equivalents thereof known to those skilled in the art and so forth . unless defined otherwise , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs . specific methods , devices , and materials are described , although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention . all publications cited herein are hereby incorporated by reference including all journal articles , books , manuals , published patent applications , and issued patents . in addition , the meaning of certain terms and phrases employed in the specification , examples , and appended claims are provided . the definitions are not meant to be limiting in nature and serve to provide a clearer understanding of certain aspects of the present invention . prostate cancer remains the second leading cause of cancer death among us men due to a subset of cancers that progress to metastatic disease . we hypothesized that germline variants in the aspartic acid ( d ) repeat domain of asporin ( aspn ) may be associated with metastatic prostate cancer progression . this study analyzed aspn d repeat domain length in a cohort of 912 patients treated with radical prostatectomy for prostate cancer at jhu . multivariable analyses demonstrated that men with an aspn d repeat domain length of 13 in both alleles ( 13 / 13 ) were significantly less likely to progress to metastatic disease ( hazard ratio [ hr ]: 0 . 43 , p = 0 . 038 ) than men with other common allelic genotypes . in contrast , multivariable analyses demonstrated that men who have an aspn d repeat domain length of 13 in one allele and 14 in the other allele ( 13 / 14 ) or who have at least one allele with 14 d repeats ( 14 / x ) were significantly more likely to have lymph node metastases at surgery ( odds ratio [ or ] for 13 / 14 : 2 . 62 , p = 0 . 006 and or for 14 / x : 1 . 95 , p = 0 . 033 ) than men with other common allelic genotypes . while homozygosity for the aspn d13 variant is significantly prognostic of a reduced risk of metastatic recurrence , heterozygous aspn d13 / 14 is significantly associated with an increased risk of lymph node metastases at surgery . these are the first germline variants reported that differentiate between indolent and aggressive prostate cancer . not only do these findings have potential clinical utility for active surveillance decisions , they also provide mechanistic insights into prostate cancer progression . the study population is a hospital - based case population at the johns hopkins hospital ( jhh ). all participants provided written informed consent . the protocol and consent documents were approved by the jhu institutional review board . prostate cancer cases were 100 % men of non - hispanic european descent ( by self report ) who underwent radical prostatectomy for treatment of prostate cancer at jhh from february 1993 through november 2012 . patients treated prior to the psa era ( 1992 and prior ) and those who received neoadjuvant hormonal treatments were excluded from the study . considering the aforementioned criteria , 1000 cases were selected at random for genomic analysis , of which 912 had adequate dna for genotyping . radical prostatectomy specimens were processed as previously described ( 20 ). each tumor was graded using the gleason scoring system and staged using the tmn ( tumor - node - metastasis ) system . patients who met any of the following criteria were considered as having more aggressive disease : pathologic gleason score of 7 or higher , stage pt3 or higher , n +, or m1 ( 21 ). clinical outcome data included biochemical recurrence ( bcr ) ( defined as a post - operative prostate specific antigen ( psa )≧ 0 . 2 ng / ml ), distant metastasis ( defined as clinical or radiographic spread of disease to extra - pelvic lymph nodes , bones , or viscera ), and prostate - cancer specific mortality ( pcsm ). tissue for genomic dna isolation was taken from seminal vesicles ( sv ) at the time of radical prostatectomy . sv tissue was suspended in 12 ml suspension buffer ( 20 mm tris ; 25 mm edta ; 100 mm nacl )+ 1 ml ?% sds + 60 μl proteinase k solution ( 20 mg / ml ), inverted twice , and then incubated overnight at 50 ° c . the next day , rna was digested by adding 60 μl rnase a solution ( qiagen ) and incubating at 37 ° c . for 1 hour . proteins were precipitated in 4 ml protein precipitation solution ( promega ) on ice for 20 minutes and then centrifuged at 2 , 000 g for 10 minutes . dna was extracted from the supernatant with 30 ml 100 % isopropanol , centrifuged 2 , 000 g for 5 minutes , and then washed with 70 % ethanol followed by centrifugation . the d repeat polymorphism located in the n - terminal region of the aspn gene was polymerase chain amplified ( pcr ) amplified using 5 ′ primer 6 - fam - attcctggctttgtgctctg ( seq id no : 1 ) and 3 ′ primer tggcttcttggctctcttgt ( seq id no : 2 ). primers were designed using oligo software . other primers can be designed using methods and software known to those of ordinary skill in the art . reactions were carried out in 10 μl consisting of 30 ng dna , 0 . 125 μm primers , 0 . 6 mm dntps ( continental lab products ), 10 mm tris - hcl ph8 . 3 , 50 mm kcl , 1 . 5 mm mgcl , and 0 . 6 units of taq gold dna polymerase ( perkin elmer ). amplification was performed in a veriti thermal cycler ( applied biosystems inc .) for an initial denaturation of 12 minutes at 94 ° c . followed by 40 cycles of 94 ° c . for 20 sec , 58 ° c . for 20 sec , 72 ° c . for 30 sec , and a final 10 minute elongation at 72 ° c . the pcr products were electrophoresed on an abi 3730xl dna analyzer ( applied biosystems inc .). data was collected and analyzed with genemapper software ( applied biosystems inc .) that calculates fragment length in reference to an internal lane standard ( liz500 ). three homozygous samples of different repeat length were confirmed by sanger sequencing . pcr products were sequenced using fluorescent dideoxy terminator method of cycle sequencing . reactions were run on a 3730xl dna analyzer ( applied biosystems division ) following applied biosystems protocols . sequence data was analyzed using sequencer software ( gene codes ). characteristics of patient groups defined by distinct aspn variations were compared . means of continuous variables were compared by t - tests . medians of non - normally distributed variables were compared by wilcoxon - mann - whitney rank - sum tests . proportions were compared by t - tests . unviariate and multivariable logistic regression analysis was performed in order to test strengths of association with aspn variations and pathologic outcomes : organ - confined disease , extracapsular extension , seminal vesical invasion , and lymph node metastasis . similarly , univariate and multivariable cox proportional hazards models were used to test the associations of aspn variations with risk of subsequent oncologic outcomes : bcr , metastasis , and pcsm . survival estimates were derived from kaplan - meier life tables . statistics were computed with stata 11 . 0 ( statacorp ). the study population included 912 men who underwent radical prostatectomy and pelvic lymphadenectomy for clinically localized prostate cancer at jhu . the median age at radical prostatectomy for treatment of prostate cancer was 59 . 0 years , with an average of 2 . 0 years follow up time after surgery ( fig3 ). to eliminate any confounding variables due to racial differences in allelic length distribution , all participants were caucasian . most men ( 93 . 5 %) had organ confined prostate cancer at surgery and the median psa level was 5 . 5 ng / ml ( fig3 ). during follow - up , 29 . 9 % of men developed biochemical recurrence , 11 . 4 % of men developed metastatic lesions , and 5 . 0 % of men died of prostate cancer ( fig3 ). men in this study had aspnd repeat lengths ranging from 10 to 19 aspartate residues with the most common lengths of 13 ( 45 . 8 %), 15 ( 22 . 2 %), 14 ( 14 . 9 %), 16 ( 7 . 2 %), and 12 ( 6 . 0 %) residues . the least common aspartate residue lengths of 10 , 11 , 17 , 18 , and 19 were each less than 2 % of the total ( fig1 ). this distribution accurately reflects that found in non - osteoarthritis control cohorts of us , uk , and spanish caucasian men and women as describe in atif et al ., 2008 ( 22 ), mustafa et al ., 2005 ( 23 ) and rodriguez - lopez et al ., 2006 ( 24 ), respectively ; suggesting that variations in aspn d repeat length are not associated with prostate cancer incidence . pre - surgical nomagrams including psa , biopsy gleason grade and clinical stage are used to estimate the probabilities of pathological stage at surgery ( 25 ) and biochemical recurrence following surgery ( 26 ). thus we examined for an association between aspn d repeat length and pre - surgical factors . the most common aspn d length genotypes in this cohort were 13 / 13 ( 22 . 0 %), 13 / 15 ( 19 . 1 %), 13 / 14 ( 13 . 6 %), and 14 / 15 ( 7 . 5 %) with the remaining genotypes between 0 . 1 % and 5 . 7 % of the study population ( fig4 ). to allow for adequately - powered comparative tests , genotypes present in ≧ 5 % of the study population were selected for further analysis ( where the integer corresponds to the number of aspartate - coding repeats in a single allele of aspn ): 13 / 13 , 13 / 14 , 13 / 15 , and 14 / 15 . furthermore , single repeat length alleles present in ≧ 10 % of the study population were also analyzed : any 13 , any 14 , and any 15 . when compared to pre - surgical variables , none of the genotypes examined were associated with biopsy gleason grade , psa or clinical stage , suggesting that aspn d length variants are not differentially associated with localized tumor growth or loss of differentiation ( fig5 ). post - surgical nomagrams including psa , gleason score and pathological stage are also used to estimate biochemical recurrence following surgery ( 26 ). similar to pre - operative gleason score from biopsy , aspn d length variants were also not associated with pathological gleason grade at surgery . aspn d length variants were also not associated with local invasion through the prostatic capsule ( extracapsular extension [ ece ]) or to the seminal vesicles ( seminal vesicle invasion [ svi ]). in addition , none of the aspn d repeat length variants were significantly associated with biochemical recurrence following surgery . in contrast to localized invasion and biochemical recurrence , two common allelic variants were significantly associated , one negatively and one positively , with metastatic tumor progression . multivariable cox regression analyses adjusted for pre - operative variables demonstrated that aspn d13 / 13 was significantly associated with a reduced risk of metastatic progression following surgery ( hr : 0 . 45 ; 95 % ci ; p = 0 . 038 ) ( fig6 ). when adjusted for post - operative variables , aspn d13 / 13 was still associated with a reduced risk of metastatic progression but lost significance ( fig6 ). kaplan - meier survival estimates support that homozygous aspn d repeat length of 13 is a significant germline marker prognostic of survival from metastatic prostate cancer ( fig2 ). aspn d13 / 13 is associated with a medium time to progression of xxx years compared to xxx years for other allelic lengths . this study supports that homozygous carriers of aspn d repeat length of 13 are less than half as likely to have metastatic prostate cancer recurrence following surgery than men with other common allelic d repeat lengths . heterozygous carriers of aspn d13 , however , are not protected from metastatic progression suggesting that aspn d13 is recessive to other allelic d repeat length variants . while homozygous asp n d13 is protective of metastatic prostate cancer recurrence , aspn d14 is associated with metastatic progression . multivariable cox regression analyses adjusted for pre - operative variables demonstrated that men with aspn d13 / 14 or carrying any aspn d repeat length of 14 ( homozygous or heterozygous ) were significantly more likely to have lymph node metastases at surgery ( or for 13 / 14 : 2 . 62 ; 95 % ci , p = 0 . 006 and or for 14 / x : 1 . 95 ; 95 % ci , p = 0 . 033 ) ( fig6 ). kaplan - meier survival estimates demonstrate heterozygous aspn d13 / 14 is a significant germline prognostic marker of metastatic prostate cancer recurrence ( fig2 ). for metastatic disease , aspn d13 / 14 was associated with a medium time to progression of xx years compared to men with other aspn d genotypes . these data suggest that men with aspn d13 / 14 are approximately twice as likely to have metastatic disease at surgery as men with other common aspn d repeat lengths . over a third of caucasian men are carriers of either homozygous aspn d13 or heterozygous aspn d13 / 14 . when compared to aspn d13 / 13 , aspn d13 / 14 was not associated with increases in psa , biopsy gleason grade , clinical stage , pathological gleason grade , ece or svi ( fig7 ). however , multivariable analyses demonstrated that carriers of aspn d13 / 14 were over three times as likely to have lymph node metastases at surgery ( or 3 . 16 , p = 0 . 008 ) and more than three and a half times as likely to have metastatic recurrence ( hr 3 . 54 , p = 0 . 006 ) than men homozygous for aspn d13 ( fig8 ). targeted analysis of germline aspn d repeat domain length was analyzed in a cohort of patients treated with radical prostatectomy for prostate cancer at jhh . these analyses demonstrated that homozygous aspn d13 / 13 is significantly protective of metastatic prostate cancer following surgery , while aspn d14 ( either homozygous or heterozygous ) is significantly prognostic of lymph node metastases at surgery . interestingly , heterozygous carriers of aspn d13 / x are not protected from prostate cancer metastases suggesting that potential protective functions of aspn d13 are recessive to other alleles , especially aspn d14 . these findings have potential to impact therapeutic decisions in the clinic . controversy exists pertaining to the overtreatment of men with very low and low risk prostate cancer . due to the indolent nature of these cancers , active surveillance is a treatment option for men with very low and low risk prostate cancer as defined in the nccn guidelines ( nccn guidelines prostate cancer version 2 . 2013 ). despite this conservative option , many patients with very low to low risk prostate cancer undergo surgical or radiotherapeutic intervention , which are not without associated side effects and costs . concerns over diagnostic accuracy of cancer aggressiveness often influence decisions towards definitive curative therapy . the development of a minimally - invasive and cost - effective genomic test for the early identification of aggressive prostate cancer has the potential to better identify those who do indeed need therapeutic intervention and as a corollary to spare thousands of men with indolent disease unnecessary surgical treatment . significant progress has been made in the past several years to identify genetic risk factors for prostate cancer . genome wide association studies ( gwas ) have identified several single nucleotide polymorphisms ( snp ) associated with prostate cancer incidence ( 27 - 35 ); yet inherited determinants of aggressive prostate cancer have remained elusive . in this study , we demonstrate that while aspn d repeat length variants do not affect the risk of being diagnosed with prostate cancer , two common variants affect the risk of having aggressive disease . while further studies and prospective trials are needed , this initial study suggests that inclusion of a genetic test for aspn d repeat length may have clinical utility for men considering active surveillance as a treatment option . due to its significantly protective association , homozygous aspn d13 carriers may be better candidates for active surveillance than men with other allelic length variants . in contrast , due to its significant association with metastatic disease , carriers of aspn d14 ( either homozygous or heterozygous ) may be better candidates for surgical intervention and possibly multi - modal therapies . as both polymorphisms in the d repeat domain length are common in caucasian men , inclusion of these criteria for better delineation between indolent and aggressive prostate cancer has the potential to impact a substantial fraction of men considering active surveillance . aspn is an extracellular matrix ( ecm ) protein that has been shown to be elevated during androgen - induced early prostate development ( 1 ) and in cancer associated fibroblasts ( 8 ). both its stromal specific expression in prostate cancer and its extracellular localization suggest that aspn may have a role in modifying the ecm environment . one could postulate that the aspn d13 allelic variant due to divergent allelic associations with metastatic prostate cancer , aspn variants may differentially regulate processes driving metastatic progression . several studies suggest that aspn may play an important role in modifying the ecm environment . the aspn d14 polymorphism is associated with susceptibility to bone and joint diseases in asian populations including osteoarthritis ( 12 , 13 , 15 , 16 ), lumbar - disc degeneration ( 17 ), and developmental dysplasia of the hip ( 18 ). this , combined with our finding that aspn d14 is also associated with metastatic prostate cancer , suggests that aspn d polymorphisms may differentially regulate pathways common to both degenerative diseases and metastatic progression . it has been postulated that aspn and in particular aspn d14 inhibits tgfβ1 mediated chondrocyte differentiation and cartilage ecm formation ( 13 ). while the d repeat domain is not necessary for tgfβ1 binding ( 36 ), polymorphisms in the length of the d repeat domain have been shown to differentially affect the ability of aspn to inhibit in vitro tgfβ1 induced cartilage matrix gene expression . aspn d14 has enhanced inhibition of tgfβ1 - induced signaling over aspn d13 while aspn d16 and d17 variants do not inhibit tgfβ1 signaling ( 13 ). in addition to tgfβ1 , aspn has also been shown to inhibit other tgf family members such as bmp2 ( 37 ); yet the role of the aspn aspartate domain length variants in bmp2 inhibition is not known . polymorphisms in the aspartate repeat domain may differentially confer susceptibility to tgf family member mediated diseases , including prostate cancer . however , a role for aspn in tgfβ1 or bmp2 signaling in the prostate is not known . in addition to functioning through tgfβ , aspn may directly modify the ecm through collagen interactions . aspn has recently been shown to regulate osteoblast - driven collagen mineralization ( 19 ). while the aspn n - terminal d repeat domain has been shown to bind calcium ( 19 ), the c - terminal domain of aspn containing 10 leucine rich repeats has been shown to bind to type i ( 19 ) and to type ii ( 36 ) collagens . aspn competes with decorin , an inhibitor of prostate tumor growth ( 38 ), for collagen binding ( 19 , 39 , 40 ). in contrast to aspn expression which is increased ( 8 ), decorin expression is decreased in prostate cancer associated fibroblasts ( 41 ). aspn and decorin may work antagonistically to regulate the ecm environment . the differential roles of aspn d length variants play in these processes have not been examined . furthermore , the biologic and molecular mechanisms by which aspn d14 may promote and aspn d13 / 13 may inhibit metastatic prostate cancer are not known . 1 . schaeffer e m , marchionni l , huang z , et al . androgen - induced programs for prostate epithelial growth and invasion arise in embryogenesis and are reactivated in cancer . oncogene 2008 ; 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