Accurate Predition of Repeat Prostated Biospys Outcomes_Robinson_Prostate Cancer Prostatic Dis_2010

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Prostate Cancer and Prostatic Diseases (2010) 13, 126–131 & 2010 Nature Publishing Group All rights reserved 1365-7852/10 www.nature.com/pcan ORIGINAL ARTICLE Accurate prediction of repeat prostate biopsy outcomes by a mitochondrial DNA deletion assay K Robinson1, J Creed1, B Reguly1, C Powell1, R Wittock1, D Klein1, A Maggrah1, L Klotz2, RL Parr1 and GD Dakubo1 1 Genesis Genomics, Thunder Bay, ON, Canada and 2Division of Urology, Department of Surgery, Sunnybrook Health Sciences Centre, Uni
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  ORIGINAL ARTICLE Accurate prediction of repeat prostate biopsy outcomes bya mitochondrial DNA deletion assay K Robinson 1 , J Creed 1 , B Reguly 1 , C Powell 1 , R Wittock 1 , D Klein 1 , A Maggrah 1 , L Klotz 2 , RL Parr 1 andGD Dakubo 1 1 Genesis Genomics, Thunder Bay, ON, Canada and 2 Division of Urology, Department of Surgery, Sunnybrook Health SciencesCentre, University of Toronto, Toronto, ON, Canada Several cancers are characterized by large-scale mtDNA deletions. We previously provided evidencethat one of these deletions has potential utility in resolving false from true-negative prostate needlebiopsies. This study was to assess the clinical value of this deletion in predicting re-biopsy outcomes.We used a quantitative polymerase chain reaction assay to measure the levels of the deletion inindividual negative needle biopsies from 101 patients who had a repeat biopsy within a year withknown outcomes. Using an empirically established cycle threshold (Ct) cutoff of 31, and the lowestCt for each patient as diagnostic of prostate cancer, as well as the histopathologic diagnosis onsecond biopsy, we calculated the clinical performance of the deletion. The Ct cutoff at 31 gave asensitivity and specificity of 84 and 54%, respectively, with the area under a receiver-operatingcharacteristics curve of 0.749. The negative predictive value was 91%. The assay was able to predictthe presence of a missed tumor in 17 out of 20 men a year before diagnosis. This ancillary testappears to identify men who do not require a repeat biopsy with a high degree of certainty. Theresults suggest that the majority of men with atypical small acinar proliferation have a concurrentmissed tumor and therefore require close monitoring for early detection. Prostate Cancer and Prostatic Diseases (2010) 13, 126–131; doi:10.1038/pcan.2009.64; published online 19 January 2010 Keywords: Negative prostate needle biopsies; mtDNA deletion; negative predictive value; fieldcancerization Introduction In the developed world, prostate cancer (PCa) remainsthe most frequent noncutaneous malignancy diagnosedin men over the age of 50 years; however, the case fatalityof PCa is lower than many cancers. The decreasedmortality is attributable to early detection of curabletumors and the slow natural history of many PCas. Earlydetection is achieved by increased awareness coupledwith diligent digital rectal examination and PSA screen-ing regimes. Although the PSA assay continues to beimportant in PCa management, it is quite inaccurate as ascreening test. Several nonmalignant conditions includ-ing BPH and prostate infections can cause elevated PSA. 1 The positive predictive value of an elevated PSA is35%. Between 15 and 30% of men with negative biopsiesharbor PCa. In these cases, thenegative results areaccounted for by sampling error. 2–5 Men whose biopsiesshow high-grade prostatic intraepithelial neoplasia(HGPIN) or atypical small acinar proliferation (ASAP)have a 24–41% risk of underlying PCa that is identifiedon repeat biopsy. 6 Cancer has been reported in up to100% of ASAP cases. 7 These findings highlight theneed to re-biopsy patients with ASAP/HGPIN within6–12 months.Men with an elevated PSA are at increased risk forPCa. A single negative biopsy is not sufficient to excludethe diagnosis with certainty. Progressive rise in the PSAafter an initial negative biopsy causes further anxiety andis often considered an indication for requiring a second biopsy. 8 An ancillary diagnostic test to rule out cancerwithout a repeat biopsy would have many advantages.We previously reported the potential clinical valueof a 3.4kb mitochondrial DNA deletion that shows theconcept of field effect or cancerization. 9 This couldresolve negative prostate biopsies into true- and false-negative categories. However, of the 96 patients in thevalidation study that provided proximal biopsy samples,only 48 had accompanying malignant biopsy cores forcomparative analysis of test performance. Though manyof these patients (30) had only a single biopsy, the 3.4kbdeletion was accurate in predicting the presence of amalignant focus in 67% of the patients. However, theperformance of the deletion improved to 78% when weexamined 18 patients who had two or more previoussamples. This suggests that the marker could predict the Received 10 September 2009; revised 4 December 2009; accepted 18December 2009; published online 19 January 2010Correspondence: Dr GD Dakubo, Genesis Genomics, 1000-290 MunroStreet, Thunder Bay, ON P7A 7T1, Canada.E-mail:gabriel.dakubo@genesisgenomics.com Prostate Cancer and Prostatic Diseases (2010) 13, 126–131 & 2010 Nature Publishing Group All rights reserved 1365-7852/10 www.nature.com/pcan  outcome for the patient with a rising PSA andnega-tive initial biopsies with a high degree of accuracy. 9 Thisstudy was designed to establish the 3.4-kb deletion biomarker cutoff that had the highest predictive accuracyfor the results of subsequent biopsies. The findingsshould form the basis for the subsequent evaluation of this marker on prostate re-biopsy outcomes. Materials and methods Patients and samples All patients were consented and samples obtained inaccordance with the ethical guidelines of the TrafalgarEthics Board (Oakville, Canada) that operates in accor-dance with the Tri-Council Policy Statement on EthicalConduct of Research Involving Humans.A total of 101 patients who have had a follow-up biopsy within 1 year of an initial negative biopsy forsuspected PCa were recruited for the study. Sections(20 m m) from the six representative anatomic regions of the prostate (right apex, right mid, right base, left apex,left mid, left base) of the initial negative biopsies wereobtained. It should be noted that by conventionalmodern practice, each patient received between 10 and12 core biopsies; however, for logistic reasons we testedonly cores representing six anatomic areas of theprostate. In all, a total of 595 (instead of 606) biopsieswere analyzed. Eleven patients provided 5 cores each(instead of the 6 requested cores). The biopsies spana period between 1997 and 2008. On second biopsy,20 patients were found to have cancer, with the remain-ing found to be benign. Ten of the PCa patients subse-quently had prostatectomies for treatment of PCa. DNA extraction and target amplification DNA was extracted from 20 m m prostate needle biopsysections using a QIAamp DNA Mini Kit (Qiagen,Mississauga, Ontario, Canada). Negative extraction con-trols were included and were monitored for amplifica-tion. Samples were quantified, diluted to 2ng m l À 1 anddistributed to master template 96-well plates. Polymerasechain reaction (PCR) amplification was performed withPerfeCta Sybr Green Supermix (Quanta Biosciences,Gaithersburg, MD, USA). The following reaction condi-tions were used: 1 Â PerfeCta Sybr Green Supermix,0.25 m M forward primer, 0.25 m M reverse primer (primersequences available upon request), 20ng template DNAin a 25 m l reaction volume. PCR was performed using thefollowing cycling parameters: 95 1 C for 2min to activatethe polymerase, followed by 45 cycles of 95 1 C for 30s,30s annealing at optimized primer temperatures: 61.5 1 Cfor total mtDNA and tumor necrosis factor, 64.4 1 Cfor deletion-specific primer and extension at 72 1 C for30s. Plate reads were performed after each extensioncycle. A 10-min final extension at 72 1 C was performed before the melting curve analysis (70–95 1 C, readingevery 1 1 C and holding for 3s). The Opticon 2 andChromo4 Real-Time Detector Systems (Bio-Rad Labora-tories, Mississauga, Ontario, Canada) were used forPCR. One negative PCR control was included on everyamplification plate for each primer set as well as sevenstandards for normalization purposes. These standardswere serial dilutions of the target amplicon generatedwith conventional PCR, purified, quantified and dilutedsuch that the seven standards were at concentrationsof 3.85ng, 0.385ng, 38.5pg, 3.85pg, 0.385pg, 38.5fg and3.85fg. Analysis of any given plate required a minimumof four standards. Statistical analysis The cycle threshold (Ct) of the 3.4kb deletion wascompared to the Ct of total amount of mtDNA (12SrRNA gene) and the amount of nuclear DNA (tumornecrosis factor). A tumor necrosis factor Ct of  4 40 for asample was considered a failed reaction because thisreflected insufficient template input. This restrictionresulted in seven failed patient samples, which wereremoved from further analysis. Template DNA (10ng)was seeded into each well for the quantitative PCR assay.Because tumor necrosis factor measured template input,the Ct variance across all samples was within 1 cycle;hence further analysis relied solely on 3.4kb deletion Ct.The mean and standard errors were calculated for eachtissue classification. Two-by-two tables were constructedfrom the Cts for cancer and benign outcomes, and usedto compute assay performance. Similarly, with referenceto second biopsy outcomes, receiver-operating character-istics (ROC) curves were constructed for deletion Cts andthe area under the curves was computed. All the testswere two-sided and statistical significance was consid-ered at a P -value o 0.05. SPSS version 13 (SPSS, Chicago,IL, USA) was used for all statistical analyses. Results The clinical and demographic data for all 101 subjects aresummarized inTable 1, and the pathological charac-teristics of the malignant patients are found inTable 2.The mean ( ± s.d.) age, PSA and duration between biopsies for the participants were 60.64 ( ± 7.99) years,7.09 ( ± 4.93) ngml À 1 and 7.72 ( ± 5.27) months, respec-tively. The group with ASAP was significantly youngerthan other patients ( P ¼ 0.05). On the initial biopsy(tested in this assay), 8 patients had no histopathologicfindings in any of their biopsies, 15 had isolated ASAP,28 had isolated PIN, 7 had both ASAP and PIN, and 43had findings such as hyperplasia, atrophy and prostaticinflammation. The majority of patients with subsequentdiagnosis of cancer appear to have limited cancer ( o 50%core involvement, Gleason score o 7 and o 3 coresinvolved, seeTable 2). Seven patients were excludedfrom further analysis because their samples failed tomeet assay quality standards. Hence, the final dataoutput based on 94 patients is composed of 20 malignantand 74 benign biopsies.Because the Cts of our previous assays were generallyhigh, 9 we optimized the assay to improve efficiency andspecificity. We redesigned the primers for the deletionand this dropped the annealing temperature from 66 to64.4 1 C. Importantly, in this study Sybr Green reagentswere used that have been specifically optimized forefficiency and sensitivity. All of these changes culmi-nated in efficient deletion target amplification. Theefficiencies for all runs in this assay were tight and mtDNA assay for making prostate re-biopsy decision K Robinson et al 127 Prostate Cancer and Prostatic Diseases  4 80% compared to the highly variable efficiencies (60–80%) from the previous study.In our previous study benign, malignant and histolo-gically normal-appearing biopsies adjacent to a malig-nant focus were tested. The greatest separation of assayscores was achieved between the malignant and benign biopsies. However, this study assayed only biopsiesshowing either benign histology, ASAP or HGPIN.Hence to establish a usable assay cutoff for accuratedelineation of benign versus malignant prostates, weused only biopsies without suspicious histological find-ings and those with a subsequent diagnosis of cancer. Weidentified 8 patients (48 core samples) that met thecriteria for benign disease and 14 malignant patients(84 samples) were selected. The mean Cts for benign andmalignant cases were 32.3 and 29.4, respectively, indicat-ing a 7.9-fold difference in the levels of the deletion between the two groups. By testing the sensitivityand specificity using an ROC curve, the optimal Ctfor accurate prediction of a positive re-biopsy outcomewas 31. Thus, the positive likelihood ratio of a missedconcurrent tumor increases or decreases with decreasingor increasing Ct, respectively (Figure 1). At this Ct cutoff,there was a statistically significant mean Ct difference between the two groups ( P ¼ 0.001). A sensitivity of 79% and a specificity of 88% were achieved. Assayperformance by ROC showed an AUC of 0.893 (95% CI;0.754–1.031) (Figure 2).The 31-Ct cutoff was used to test the outcomes of all samples in a blinded fashion. Our initial data ana-lysis revealed that a disproportionately high numberof prostates with initial or subsequent findings of ASAP were called malignant by the assay. These wereremoved and the remaining benign samples were testedseparately. The difference between the mean Cts of the benign and malignant samples was significant at P ¼ 0.001. In reference to the histopathologic diagnosison second biopsy, the assay had a sensitivity of 84%and a specificity of 54%. In men with initial negative biopsies, the false-negative rate was low and the negativepredictive value was 91%. The AUC was 0.749 (95% CI;0.625–0.873) (Figure 3).Of the 101 patients in this study, 22 had ASAP oninitial biopsy of which 10 were diagnosed with cancerwithin a year. This rate (45.5%) of cancer on re-biopsyof patients with ASAP is consistent with other reports. 10 The assay also predicted the presence of concurrentcancer in 8 of the remaining 12 subjects who had nocancer on re-biopsy. Six patients with initial negative biopsies were found to have ASAP on re-biopsy, andthe assay predicted the presence of tumor on the initial Table 2 Clinical data on patients with prostate cancer PSA (ngml À 1 ): N Gleasonscore: N Biopsyinvolved(%): N No. of coresinvolved: N No. of positive coresby assay 0–4: 1 6: 13 1–10: 10 1–2: 16 1–2: 64.1–10: 17 7: 1 10.1–50: 7 3–4: 3 3–4: 4 4 10: 2 8: 1 4 50: 1 NR: 1 5–6: 7NR: 1 NR: 2 NA: 3 (Benign) Abbreviations: NA, not applicable; NR, not recorded. 31 CYCLE THRESHOLDCUTOFF Increasing likelihood ofmissed prostate cancerIncreasing likelihood ofbenign prostate glandPositive likelihood ratioPCR cycle threshold35.033.527.029.51.01.21.93.35.4 Figure 1 A depiction of the scale used in making predictions of the likelihood of no tumor or missed concurrent tumor during the biopsy procedure. PCR, polymerase chain reaction. Table 1 Clinical and demographic characteristics of patients PCa a  ASAP a  HGPIN  a Benign a P -value No. of patients 20 10 14 57Age (years) mean ± s.d. 63.05 ± 7.46 56.22 ± 8.28 61.67 ± 8.35 60.97 ± 7.31 0.50PSA (ngml À 1 ) mean ± s.d. 6.35 ± 2.53 7.95 ± 5.41 6.83 ± 6.83 7.32 ± 5.04 0.652Time between biopsies (months) ± s.d. 7.75 ± 6.34 5.83 ± 4.19 7.21 ± 4.99 9.16 ± 5.12 0.179 Gleason grade p 6 177 2 NA NA NA NA X 8 1 % Core involved p 10 9 4 10, p 50 8 NA NA NA NA 4 50 1NA 2 Abbreviations: ASAP, atypical small acinar proliferation; HGPIN, high grade prostatic intraepithelial neoplasia; NA, not applicable; PCa, prostate cancer. a Findings on second biopsy procedure. mtDNA assay for making prostate re-biopsy decision K Robinson et al 128 Prostate Cancer and Prostatic Diseases   biopsy in all six patients. Thus, 78% of the cases withASAP on either the initial or subsequent biopsy wereflagged as having cancer by the assay (Figure 4).The extent of the field alterations in the prostatedefined by the 3.4-kb mitochondrial genome deletion isunknown. However, given that the deletion was able topredict a positive biopsy in 17 out of 20 (85%) individualswith cancer a year prior, we envisioned this as an earlyevent that might involve multiple or large areas of theprostate. To gain an insight into the possible extent of this field effect, we looked at the number of cores calledpositive by the assay in each malignant patient. Of the17 positive cases, 11 had more than 3 positive biopsies,which is suggestive of an extensive field effect (Table 2).The archived samples used for the study werecollected between 1997 and 2008 (spanning 12 years).Because both the time of fixation before embedding andage of the block adversely affect nucleic acid quality, we Area Under the Curve Test Result Variable(s): Deletion 0.8930.0710.003 0.754 1.031Area Std. ErrorAsymptoticSig.Lower Bound Upper BoundAsymptotic 95% ConfidenceI nterval p  = 0.001    S  e  n  s   i   t   i  v   i   t  y 1.000.800.600.400.200.000.00 0.20 0.40 0.60 0.80 1.0033 ab 323130Diagnosis1-SpecificityBenign Malgnant    M  e  a  n  +  -   1   S   E   D  e   l  e   t   i  o  n Figure 2 Assay performance in discriminating between patients whose initial and subsequent biopsies were benign without anyhistopathologic changes and those with malignant diagnosis on subsequent biopsies. ( a ) Mean cycle threshold (Ct) ± s.e. for the benign andmalignant groups ( P ¼ 0.001). ( b ) Receiver-operating characteristic curve for the deletions Cts calculated with reference to subsequenthistopathologic outcomes. Area Under the Curve ab Test Result Variable(s): Deletion 0.749 0.063 0.001 0.625 0.873Area Std. ErrorAsymptoticSig.Lower Bound Upper BoundAsymptotic 95% ConfidenceInterval    M  e  a  n  +  -   1   S   E   D  e   l  e   t   i  o  n 31.531.030.530.029.529.0DiagnosisBenign Malignant1- Specificity    S  e  n  s   i   t   i  v   i   t  y 1.000.800.600.400.200.000.60 0.80 1.000.400.200.00 Figure 3 Assay performance in discriminating between patients who remained benign from those diagnosed with cancer on re-biopsy.( a ) Mean cycle threshold (Ct) ± s.e. for the benign and malignant groups ( P ¼ 0.001). ( b ) Receiver-operating characteristic curve for thedeletions Cts calculated with reference to subsequent histopathologic outcomes. 31.531.030.530.029.529.028.5Group    M  e  a  n  +  -   1   S   E   D  e   l  e   t   i  o  n Benign Malignant ASAP Figure 4 Atypical small acinar proliferation (ASAP) in the prostateis classified by the assay as malignant in a majority of cases. mtDNA assay for making prostate re-biopsy decision K Robinson et al 129 Prostate Cancer and Prostatic Diseases
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