Aneusomies of Chromosomes 8 and Y Detected by Fluorescence in Situ Hybridization Are Prognostic Markers for Pathological Stage C (Pt3nom O) Prostate Carcinoma I

Vol. 2, 137-145, January 1996 Clinical Cancer Research 137

Aneusomies of Chromosomes 8 and Y Detected by Fluorescence in Situ Hybridization Are Prognostic Markers for Pathological Stage C (pT3NoM o) Prostate Carcinoma I

Satoru Takahashi, Antonio Alcaraz, (P < 0.001), respectively. These results demonstrate that James A. Brown, Thomas J. Borell, aneuploidy and specific aneusomies detected by FISH are potential markers for a poor prognosis in histological high- John F. Herath, Erik J. Bergstralh, grade pathological stage C (pT3NoMo) prostate carcinoma. Michael M. Lieber, and Robert B. Jenkins 2 Departments of Urology [S. T., A. A., J. A. B., M. M. L.] and INTRODUCTION Laboratory Medicine and Pathology [T. J. B., J. F. H., R. B. J.] and Section of Biostatistics [E. J. B.], Mayo Clinic and Foundation, Prostate adenocarcinoma has extensive variability in clin- Rochester, Minnesota 55905 ical behavior. Accurate prediction of individual tumor progression probability and patient survival is a major goal of current prostate cancer research. Parameters such as clinical and patho- ABSTRACT logical stage, histological grade, and pretreatment serum PSA 3 In an attempt to identify new prognostic markers, we are conventionally used to help predict the prognosis for indi- performed fluorescence in situ hybridization (FISH) ploidy vidual patients with clinically localized prostate carcinoma (1). analysis of tumor tissue from patients with a targeted stage Newer factors, such as DNA content ploidy analysis using FCM and histological grade of prostate carcinoma. We identified or static image analysis, can help refine prognostic risks (2). all 227 patients from the Mayo Clinic radical prostatectomy However, it is recognized that FCM or static image ploidy data base who had a high histological grade pathological analysis cannot detect small changes in DNA content or chro- stage C (PT3NoM o) tumor removed between 1966 and 1987. mosome number. After histological review of the paraffin-embedded specimen Interphase cytogenetic analysis FISH, using chromosome blocks, 181 cases were suitable for FISH analysis using enumeration probes, is useful for detecting numerical chromo- chromosome enumeration probes for chromosomes 7, 8, 10, some alterations in a variety of solid tumors (3, 4). Previous 12, X, and Y. FISH detected 80 (44%) diploid, 22 (12%) studies from this laboratory have demonstrated that FISH anal- tetraploid, and 79 (44%) aneuploid tumors. The common ysis is more sensitive than FCM for detecting aneuploidy of aneusomies were of chromosomes 7 and 8, which were radical prostatectomy specimens, and that aneusomies of chro- present in 51 (28%) and 46 (25%) tumors, respectively. mosomes 7 and 8 are associated with higher histological grade Aneusomies of chromosomes 10, 12, X, and Y were ob- and advanced pathological stage (5-7). These results encour- served in 11 (6%), 15 (8%), 12 (7%), and 16 (9%) tumors, aged us to evaluate the hypothesis that nonrandom numerical respectively. chromosome alterations detected by FISH are useful markers for FISH aneuploid tumors showed a trend of more fre- tumor progression and patient prognosis. As a preliminary trial, quent systemic prostate cancer progression than nonaneu- we performed a case-control FISH study using two groups of 25 ploid tumors (P = 0.060). For individual chromosome anom- patients with clinically localized prostate carcinoma who were alies, gains of chromosome 8, aneusomy of chromosome 8, matched for clinicopathological features but who showed dis- and aneusomy of chromosome Y correlated highly with tinctively different prognoses (8). This study demonstrated that systemic cancer progression (P = 0.006, 0.013, and 0.021, overall aneuploidy and aneusomy of chromosome 7 were sig- respectively). Gains of chromosome Y and aneusomy of nificantly associated with relatively rapid prostate cancer death. chromosome Y were associated with an increased prostate We now have performed an inclusive retrospective cancer death rate (P < 0.001 for both). Multivariate analysis FISH tumor analysis of patients with a single defined patho- showed that gains of chromosome 8 and aneusomy of chro- logical stage and histological grade of prostate adenocarci- mosome Y were significant independent "predictors" of noma. We targeted histological high-grade pathological stage systemic cancer progression (P = 0.008) and cancer death C (pT3NoM o) prostate cancers treated by radical prostatectomy. Clinical tumor progression rates and cause-specific survival of the patients with this grade and stage of prostate carcinoma are difficult to predict. In an attempt to identify useful new prognostic markers for these cancers, FISH analysis Received 5/9/95; revised 7/21/95; accepted 8/15/95. 1 This work was supported by a Specialized Project of Research Excel- was carried out using chromosome enumeration probes for six lence P20 Grant from the National Cancer Institute (CA 58225; to different chromosomes. R. B. J. and M. M. L.), by a grant from Vysis, Inc. (to R. B. J.), and by the Hospital Clinic of Barcelona and Fondo de Investigaciones Sanitar- ias (FIS 93/5326; to A. A.) 2 To whom requests for reprints should be addressed, at Cytogenetics Laboratory, Mayo Clinic, 200 First Street S.W., Rochester, MN 55905. 3 The abbreviations used are: PSA, prostate-specific antigen; FCM, flow Phone: (507) 284-4696; Fax: (507) 284-0043. cytometry; FISH, fluorescence in situ hybridization.

MATERIALS AND METHODS chromosomes 4, 6, 7, 8, 9, 10, 11, 12, 17, 18, X, and Y (6, 8). Study Design. Of 1786 patients in the Mayo Clinic rad- The FISH procedure was carried out in the same manner as ical prostatectomy data base whose tumors were surgically described previously (8). In brief, slides with isolated nuclei removed between 1966 and 1987, 637 (36%) were found to have were dehydrated in an ethanol series (70, 85, and 100%). DNA pathological stage C (pT3NoMo) prostate cancer. All patients was denatured by incubating the slides in 70% formamide/2X had pelvic lymphadenectomies concurrently and did not have SSC at 75°C for 4 min followed by dehydration in an ice-cold metastatic deposits in the pelvic lymph nodes. Pathological ethanol series. Simultaneously, a dual-color probe solution (7 ~1 stage C (PT3) disease denotes prostate cancers that have perfo- Hybridization Mix II; Vysis, and 2 p~l of each probe) was rated the prostatic capsule and involve periprostatic tissues, denatured for 6 rain at 75°C and subsequently added to the slides. After sealing, the slides were then incubated overnight at seminal vesicles, membranous urethra, and bladder neck (1). 37°C. Following hybridization, the slides were washed in 50% Among this large group, 227 patients who had histological formamide/2X SSC, 2x SSC, and 2XSSC/NP40. Nuclei were high-grade tumors and adequate clinical follow-up were se- counterstained with 1 ~g/ml 4',6-diamidino-2-phenylindole di- lected as our study group. Tumor grade was determined by the hydrochloride. Mayo nuclear morphological grading system, and tumors of The FISH signals were counted with a Zeiss Axioplan grade 3 or 4 were considered "high-grade tumors" for this microscope equipped with single-pass filters (150191 and study (9). 150291; Vysis). The hybridization signals for each probe in 300 The clinicopathological data available for these patients interphase nuclei were counted. To minimize sampling error, included patient age, tumor volume, seminal vesicle involve- two individuals counted signals in 150 nuclei on each half of the ment (if present), preoperative serum PSA level (which became hybridized area. All morphologically intact nuclei (apparently available in 1987), flow cytometric DNA ploidy, and adjuvant truncated or overlapping nuclei were excluded) within that area therapy (if applied). Tumor volume was calculated as described were evaluated. When an obvious discrepancy was observed preyiously (10), and serum PSA level was measured with a between these independent counts by two individuals, a third monoclonal solid-phase two-site immunoradiometric assay (Hy- individual enumerated signals on the same slide without infor- britech, Inc., San Diego, CA). FCM analysis on formalin-fixed mation of the previous results, and the two most concordant paraffin-embedded prostatectomy specimens was performed results of the three were used for analysis. The total percentage with the same method as previously described (5). Systemic of nuclei containing one, two, three, four, five, six, seven, eight, prostate cancer progression and prostate cancer death were used and more than eight signals was determined for each chromo- as clinical end points. Systemic progression was defined as some. clinical evidence of distant metastatic disease and ascertained by Criteria for FISH Ploidy and Aneusomy. To define positive findings on bone scan or other radiological imaging ploidy and aneusomy objectively, previously defined criteria tests. were used (6-8). In brief, based upon the mean + 3 SDs of The patient list was randomized, and the following tumor centromere copy numbers in benign prostate hyperplasia sam- specimen FISH analyses were performed without knowledge of ples and an inspection of prior prostate carcinoma FISH results, the clinicopathological findings and survival data of the patients. the following conservative cutoff values were established. Tissue Preparations. For each case, a surgical patholo- 1. A tumor was classified as tetraploid if the autosomal gist previously had identified a single prostate specimen block average of the percentage of nuclei with four signals was ->6%. which contained the highest histological grade prostate cancer 2. An abnormal monosomy (or nullisomy for sex chromo- for FCM ploidy analysis. These paraffin-embedded tumor somes) required ->12% of the nuclei to contain one (or zero) blocks were sectioned and histologically reviewed, and only FISH signal. those specimens with ->30% cancer cells were used in this 3. An abnormal autosomal trisomy was required to fulfill investigation. After review, 181 cases were suitable for FISH two criteria: (a) ->7% nuclei contain three FISH signals and (b) analysis. Three adjacent 50-1xm tumor tissue sections were used the ratio of three signal nuclei:four signal nuclei be >-2:3. The for FISH analyses. Tissue deparaffinization for FISH on isolated latter criterion is necessary because in tetraploid tumors the nuclei was carried out using the previously described technique three-signal population may be elevated as a result of inefficient (8). In brief, sections were washed with 2 ml Histo-Clear (Na- hybridization. tional Diagnostics, Atlanta, GA) three times. The tissue was 4. Sex chromosome gain or tetraploidy required ->12% then dehydrated in 100% ethanol and rinsed in water. Tissues nuclei with two FISH signals. were digested in pepsin solution for 2.5 h at 37°C. After filter- 5. To distinguish sex chromosomal gain from tetraploidy, ing, isolated nuclei were washed twice with PBS and vortexed. the former required that the ratio of the percentage of nuclei The resultant nuclear suspension was applied to Superfrost with two sex chromosome signals to the average autosomal four slides, and the slides were oven dried at 65°C. signal percentage be ->2.1 FISH with Chromosome Enumeration Probes. FISH 6. Abnormal hypertetrasomy required that the sum of the probes, labeled with SpectrumOrange or SpectrumGreen, spe- percentage of nuclei with ->5 signals for any autosome or ->3 cific for the centromere region of 5 chromosomes (7, 8, 10, 12, signals for any sex chromosome be >-6%. and X) and for the mid-distal Yq (Yql2) region (Vysis, Down- 7. Abnormal monosomy/nullisomy, trisomy, or hypertetra- ers Grove, IL) were used. These probes showed acceptable somy defined a tumor as aneuploid. Otherwise, the tumor was quality of FISH signals and detected frequent numeric alter- classified as apparently diploid or apparently pure tetraploid ations in our previous studies using 12 enumeration probes for based on the overall average autosomal tetrasomy.

Table 1 Centromere copy number of representative diploid, tetraploid, and aneuploid cases Centromere copy no. (%)" Chromosome 0 1 2 3 4 ->5 Case 1 (diploid) 7 0.0 4.3 92.0 1.0 2.7 0.0 8 0.0 1.0 94.0 3.3 1.7 0.0 10 0.0 4.0 94.3 0.0 1.7 0.0 12 0.0 3.3 94.0 0.7 2.0 0.0 X 1.7 93.3 4.7 0.3 0.0 0.0 Y 1.0 93.3 5.3 0.3 0.0 0.0 Case 9 (tetraploid) 7 0.0 1.7 69.7 5.7 22.3 0.7 8 0.0 3.3 61.0 5.3 30.0 0.3 10 0.3 1.0 61.0 5.7 31.7 0.3 12 0.0 1.7 70.0 4.0 24.0 0.3 X 3.0 59.3 36.7 0.7 0.3 0.0 Y 2.7 60.3 36.7 0.3 0.0 0.0 Case 40 (aneuploid) 7 0.0 1.7 37.0 56.3 4.0 1.0 (aneusomies, +7,-8) 8 0.0 14.7 81.3 2.0 2.0 0.0 10 0.0 2.3 93.3 2.7 1.7 0.0 12 0.0 5.0 88.7 4.0 2.3 0.0 X 1.0 92.0 7.0 0.0 0.0 0.0 Y 4.0 91.7 4.3 0.0 0.0 0.0 Case 208 (aneuploid) 7 0.0 4.0 83.3 3.3 9.3 0.0 (aneusomies, +8, +X, +Y) 8 0.0 2.0 72.3 18.7 5.0 2.0 10 0.0 2.0 84.3 4.0 8.3 0.0 12 0.0 2.0 85.7 2.3 10.0 0.0 X 0.0 63.0 37.0 0.0 0.0 0.0 Y 0.0 64.3 35.3 0.3 0.0 0.0 "Percentage of 300 nuclei with indicated centromere copy number.

Table 1 shows typical examples of a FISH diploid, tet- all tests were two-sided with a level of 0.05. Survival curves raploid, and two aneuploid tumors. The FISH diploid tumor from date of prostatectomy to the end points of cause-specific (case 1) had an average autosomal tetrasomic population of 2% death and systemic cancer progression were estimated using the and, based on the criteria listed above, contained no apparent Kaplan-Meier method. Comparison of survival curves was done chromosome centromere anomaly. The FISH tetraploid tumor using the log rank test. Multivariate analyses for systemic cancer (case 9) had an average autosomal tetrasomy of 24.7% and, progression and prostate cancer death were performed using the except for the increased tetrasomy for each autosome and in- Cox proportional hazards model. Candidate predictors were age, creased disomy for each sex chromosome, contained no appar- seminal vesicle involvement, estimated tumor bulk, adjuvant ent chromosome centromere anomaly. The first FISH aneuploid therapy, FCM DNA ploidy, FISH ploidy (aneuploid versus tumor (case 40) had an average autosomal tetrasomy of 2.5% nonaneuploid), aneusomies of chromosomes 7, 8, 10, 12, X, and and, based on the criteria listed above, was observed to have Y, and gains of chromosomes 7, 8, 10, 12, X, and Y. Stepwise abnormal centromere signal distributions for chromosomes 7 backwards variable selection was used with oL level of 0.01. and 8 (aneusomies, +7 and -8). The second FISH aneuploid Preliminary evaluation of FISH ploidy with three groups tumor (case 208) had an average autosomal tetrasomy of 8.2%, found similar outcomes for diploid and tetraploid cases with which met the tetraploid criteria, but also was observed to have respect to systemic cancer progression and cause-specific death. abnormal centromere signal distributions for chromosomes 8, X, In view of this, the FISH ploidy was analyzed as aneuploid and Y (aneusomies, +8, +X, and +Y). versus nonaneuploid. A Bonferroni-type correction of the P Statistical Analysis. After evaluating all FISH signal values was done to adjust for this post hoc pooling of the data. count results and classifying FISH anomalies according to the The univariate P values from the log rank test for FISH ploidy criteria described above, their association with clinicopatholog- were multiplied times 2, the number of ways we might have ical and prognostic parameters of the patients studied were considered grouping the data (i.e., diploid versus nondiploid or analyzed statistically. Among FISH anomalies, we indepen- aneuploid versus nonaneuploid). dently tested overall FISH aneuploidy and aneusomies (pooling gains and losses) of chromosomes 7, 8, 10, 12, X, and Y against clinical end points. Gains of chromosomes 7, 8, 10, 12, X, and RESULTS Y were also examined. Chromosomal losses were not analyzed Ploidy and Numeric Chromosome Alterations Detected separately because of the small numbers of cases with this type by FISH. Table 2 summarizes the distribution of FISH results of numeric anomaly. in the 181 tumor specimens studied. According to the criteria Comparison of patient groups with respect to baseline data described above, FISH detected 80 (44%) diploid, 22 (12%) was done using the X2 or rank sum test. Unless otherwise noted, tetraploid, and 79 (44%) aneuploid tumors. The common aneu-

Table 2 Distribution of FISH results in 181 patients with histological FISH ploidy, specific chromosomal alterations (including data high-grade pathological stage C (pT3NoMo) not shown in Table 3 for chromosomes 7, 10, 12, and X), and prostate carcinoma clinicopathological parameters for this relatively homogeneous FISH findings No. of patients (%) group of patients with high-grade stage C (pT3NoMo) prostate Ploidy carcinoma. Diploid 80 (44.2) Correlation with Systemic Cancer Progression and Pa- Tetraploid 22 (12.2) tient Survival. Table 4 summarizes relationships between Aneuploid 79 (43.6) FISH results and patient prognosis. The median clinical fol- Aneusomy of chromosome Gain Loss Total low-up of the 181 patients was 6.8 (range, 0.4-21) years. 7 50 1 51 (28.2) 8 39 7 46 (25.4) During the period of follow-up, 57 (31%) patients had systemic 10 9 2 11 (6.1) prostate cancer progression. The percentage of patients without 12 15 0 15 (8.3) systemic cancer progression was 77%, 62%, and 35% at 5, 10, X 12 0 12 (6.6) and 15 years, respectively (Fig. 1A). Aneuploid cases showed a Y 12 4 16 (8.8) trend of more frequent systemic cancer progression, with a Presence of hypertetrasomy 13 (7.2) systemic progression-free probability at 10 years of 53% com- No. of aneusomic chromosomes pared to 68% for nonaneuploid cases (adjusted P = 0.060; Fig. 0 102 (56.4) 1 40 (22.1) 1B). Individual aneusomy of chromosome 8 correlated with 2 23 (12.7) more frequent systemic cancer progression (P = 0.013, Table 3 9 (5.0) 4). Moreover, cases with gain of chromosome 8 showed a 4-6 7 (3.9) stronger association with systemic progression (P = 0.006, Table 4), with a systemic progression-free rate of 44% at 10 years compared to 68% for cases without gain of chromosome 8 (Fig. 1C). Aneusomy of chromosome Y correlated with sys- somies were of chromosome 7 and 8, which were observed in 51 temic progression (P = 0.021, Table 4; Fig. 1D), but when cases (28%) and 46 (25%) tumors, respectively. Aneusomies of chro- with and without chromosome Y gain were compared, this mosomes 10, 12, X, and Y were found in 11 (6%), 15 (8%), 12 correlation no longer remained statistically significant (P -- (7%), and 16 (9%) tumors, respectively. Gains of chromosome 0.15, Table 4). relative to the inferred stemline ploidy level were more common than losses (Table 2). Thirteen (7%) aneuploid tumors showed Thirty-five (19%) patients died from prostate cancer during hypertetrasomic (->5 signals) anomalies. Forty (22%) aneuploid the period of follow-up. Overall prostate cancer-specific sur- tumors had one, 23 (13%) had two, and 16 (9%) had ->3 vival was 91%, 79%, and 49% at 5, 10, and 15 years, respec- aneusomic chromosomes. tively (Fig. 2A). Prostate cancer-specific survival of patients Correlation with Clinicopathological Characteristics. with FISH aneuploid tumors was 77% at 10 years compared to The overall mean age at surgery was 65 years. Of the 181 80% of patients with FISH nonaneuploid tumors (P :- 0.38, studied patients, seminal vesicles were invaded in 110 (61%) Table 4; Fig. 2B). Gains of chromosome 8 did not show signif- patients. Eighteen (60%) of 30 evaluable patients showed >10 icant association with prostate cancer death (P = 0.24, Table 4; ng/ml preoperative serum PSA level. Standard FCM DNA Fig. 2C). Both aneusomy and gains of chromosome Y strongly ploidy analysis of the formalin-fixed paraffin-embedded radical correlated with prostate cancer death (P < 0.001 for both; Table prostatectomy specimens was possible for 167 patients and 4). Ten-year cancer-specific survival of patients with aneusomy identified 54 (32%) diploid, 88 (53%) tetraploid, and 25 (15%) Y was 52%; in contrast, that of patients with the normal chro- aneuploid tumors. Fifty-six (31%) patients received adjuvant mosome Y number was 81% (Fig. 2D). hormonal therapy and 45 (25%) received radiotherapy. No other chromosomal aneusomies or chromosomal gains Potential associations between FISH results and the clini- correlated with systemic cancer progression or prostate cancer copathological characteristics of the patients were analyzed (Ta- death. In addition, neither the presence of hypertetrasomy nor ble 3). Aneuploid tumors had a higher percentage of elevated the number of aneusomic chromosomes correlated with these preoperative serum PSA level (>10 ng/ml) than nonaneuploid clinical end points. tumors (82% versus 47%; P = 0.063). This difference did not To examine the strength of the association of FISH anom- reach statistical significance because of a small number of cases alies with systemic cancer progression and prostate cancer death with preoperative PSA values (n = 30). Of 167 tumors success- after adjustment for the clinicopathological variables, multiva- fully studied by both FISH and FCM, 102 (61%) showed riate analysis was performed using the Cox model described in concordance of ploidy (P = 0.010). However, FISH using six "Materials and Methods." A gain of chromosome 8 was the chromosome enumeration probes was more sensitive to detect only significant independent predictor of systemic cancer pro- aneuploidy than FCM (74 aneuploid tumors detected by FISH gression among all of the examined variables (P = 0.008). versus 25 by FCM). Aneusomy of chromosome 8 was associ- Aneusomy of chromosome Y was the only significant indepen- ated with more frequent FCM aneuploidy (P = 0.018). Approx- dent predictor of prostatic cancer death (P < 0.001). imately 50% of the patients underwent an adjuvant treatment(s), and no significant differences for the percentage were observed DISCUSSION when the patient groups were stratified by FISH anomalies. No At this institution, about one-third (36%) of the patients other significant correlations were identified between overall undergoing radical prostatectomy for clinically localized pros-

Table 3 Clinicopathological characteristics and FISH results of 181 patients with histological high-grade pathological stage C (pT3NoMo) prostate carcinoma Individual aneusomya FISH ploidy Clinicopathological Normal Normal characteristics Nonaneuploidb Aneuploid U chromosome 8 Aneusomy 8 PC chromosome y Aneusomy y U Age Mean (yr) 65 64 0.26 65 64 0.28 65 64 0.28 Range 50-78 52-75 50-78 52-75 50-78 54-69 Tumor bulkd >10 cm3 % 48 63 0.066 53 56 0.77 54 56 0.92 Seminal vesicle involvement Yes (%) 59 65 0.43 59 70 0.18 61 69 0.52 Preoperative serum PSA levele >10 ng/ml (%) 47 82 0.063 58 67 0.71 59 100 0.41 FCM ploidyf Aneuploid (%) 9 23 0.010 11 26 0.018 14 29 0.14 Adjuvant therapy g Yes (%) 46 49 0.66 50 41 0.33 47 50 0.83 a Only aneusomies that have significant correlation with clinical endpoints (see Table 4) are listed. b Nonaneuploid group consists of diploid (n = 80) and tetraploid cases (n = 22). c Comparing the distribution of each parameter among FISH results using the X2 or rank sum (for age) test. d One hundred fifty cases had tumor volume available. e Thirty cases treated during 1987 were available. Only one case with aneusomy Y was available. f One hundred sixty-seven cases had FCM ploidy available. g Hormonal and/or radiotherapy within 90 days after prostatectomy.

tate cancer from 1966 to 1987 were found to have pathological than nonaneuploid tumors. Serum PSA level has been reported stage C (pT3NoM o) tumors (1 l). It is difficult to predict clinical to help predict clinical outcome (l 3). Unfortunately, PSA levels progression rates and survival for the patients with pathological were not routinely determined prior to 1987, making more stage C disease, especially those with histological high-grade detailed analysis of potential associations impossible. tumors (1). A previous study of pathological stage C patients To date, the prognostic relevance of genetic alterations on from this institution indicated that FCM ploidy provides signif- chromosome Y has not been studied in prostate cancer. In this icant additional prognostic information for patients with low study, both gains and aneusomy of chromosome Y were asso- histological grade tumors, but not for patients with high-grade ciated with an increased prostatic cancer death rate. Moreover, tumors (12). aneusomy Y was a significant independent predictor of cancer To the best of our knowledge, this is the first comprehen- death. In previous standard cytogenetic studies, loss of chromo- sive retrospective survival analysis to evaluate the prognostic some Y was reported as one of the most common clonal aber- value of numeric chromosome alterations detected by FISH in rations in prostate cancer (14, 15) and in a variety of other prostate cancer or any solid tumor. FISH analysis using chro- neoplasms including leukemia, renal cell carcinoma, and brain mosome enumeration probes was routinely applicable to archi- tumors (15-18). Loss of chromosome Y was also detected in val paraffin-embedded specimens of pathological stage C (pT3) non-neoplastic brain and kidney tissue and in bone marrow cells prostate carcinoma. The overall distribution of nuclear ploidy of healthy elderly men. Therefore, its significance as a cancer- and aneusomies found in this investigation was essentially iden- related alteration has been controversial (15, 19). However, tical to that found in our previous FISH analyses for clinically recent interphase FISH studies, including ones from our insti- localized prostate cancers (most of them are of pathological tution, have demonstrated more frequent gains of chromosome stage B or C; Refs. 5-7), except for a slightly higher frequency Y in prostate cancer (6, 8, 20, 21). This alteration is apparently of aneuploidy and aneusomies. This finding may be reasonable confined to cancerous prostatic epithelial cells (20, 21). On the because in the previous reports cases with a tumor of higher basis of these literature and our new findings, we believe that grade or advanced pathological stage showed more frequent gains as well as general aneusomy of chromosome Y is a aneusomy (6, 7), and this study targeted only high histological relatively common genetic alteration of prostate adenocarci- grade pathological stage C disease. Our previous studies also noma and may be a useful new marker for poor prognosis in demonstrated that ploidy detected by FISH is consistent with pathological stage C prostate cancer. The role of chromosome Y that detected by FCM and a degree of numerical chromosome aneusomy itself in cancer progression is not clear from the alterations corresponds well to FCM DNA index values (5-7). experiments we performed. Although the presence of aneusomy However, these studies (5-7) also demonstrated that FISH is Y correlated with progression, the presence of chromosome Y more sensitive and accurate than FCM for the detection of gain did not. This observation suggests that chromosome Y loss aneuploidy. These observations were confirmed by this inves- may be associated with progression. Indeed, three of the eight tigation. progression events observed for the 16 patients with aneusomy FISH aneuploid tumors showed a trend of more frequent Y occurred in the 4 patients with loss of the Y. However, this systemic cancer progression and higher preoperative PSA level correlation of loss of the Y with progression did not reach

Table 4 FISH results and clinical end points for 181 patients with histological high-grade pathological stage C (pT3NoMo) prostate carcinoma % (SE) of patients at 10 years a Systemic cancer Prostate cancer- FISH result progression-free pb specific survival pb Overall Ploidy Nonaneuploidc 68 (8) 80 (60) Aneuploid 53 (9) 0.060 a 77 (5) 0.38 a Individual chromosome alterations 7: Normal 59 (8) 77 (5) Aneusomic 67 (8) 0.88 85 (5) 0.50 No gain 59 (8) 77 (5) Gain 67 (8) 0.82 85 (6) 0.53 8: Normal 67 (6) 79 (5) Aneusomic 47 (11) 0.013 76 (7) 0.50 No gain 68 (6) 80 (5) Gain 44 (1 l) 0.006 73 (8) 0.24 10: Normal 65 (5) 80 (4) Aneusomic 33 (25) 0.33 66 (16) 0.46 No gain 62 (6) 80 (4) Gain 58 (19) 0.41 61 (18) 0.20 12: Normal 62 (6) 79 (5) Aneusomic (gain) e 53 (14) 0.28 79 (11) 0.75 X: Normal 61 (6) 80 (4) Aneusomic (gain) e 71 (14) 0.82 63 (15) 0.11 Y: Normal 62 (6) 81 (4) Aneusomic 51 (13) 0.021 52 (13) <0.001 No gain 62 (6) 81 (4) Gain 62 (15) 0.15 54 (16) <0.001

a Kaplan-Meier progression-free rate or survival estimate (SE) at 10 years. There were 16 patients at risk for systemic progression and 23 at risk for prostate cancer-specific death at 10 years. o p values from log rank test comparing overall progression-free rate or survival curves. c Nonaneuploid group consists of diploid (n = 80) and tetraploid tumors (n = 22). d Adjusted P value equals to two times nominal P value.

e Losses were not observed for chromosomes 12 and X.

statistical significance. It may be that aneusomy of chromosome overall survival for the patients with breast carcinoma (29). Y is simply a marker of poorer prognosis tumors and is not Furthermore, amplification of DNA sequences from 8q24 was pathogenetically related to their worse clinical behavior. recently observed in prostate cancer patients with lymph node Gains and aneusomy of chromosome 8 correlated with metastasis or recurrent disease using FISH with a region-spe- systemic cancer progression, and, moreover, gain of chromo- cific probe (30). In this study, gain of chromosome 8 correlated some 8 was a significant independent predictor for systemic with systemic cancer progression but not increased cancer death. progression. Previous FISH studies from our research group The reason for this finding is unclear. It is possible that the gain have shown that gains of chromosome 8 are common in clini- of chromosome 8 or 8q has importance for metastatic potential, cally localized prostate cancers and are associated with higher but that a subpopulation of tumor cells with this change requires tumor grades and advanced pathological stages (6, 7). Molecular additional genetic alterations to become more aggressive (e.g., genetic alterations in chromosome 8 have been implicated in the become androgen independent). carcinogenesis of multiple tumors (22, 23). Homozygous dele- Gain of chromosome 7 was the most common numeric tion and frequent allelic loss of 8p22 loci in prostate cancers chromosome alteration found in this study, but showed no have been reported, suggesting the presence of a putative tumor apparent individual prognostic relevance. In previous FISH suppressor gene in this region (24). Additionally, multiplication studies from this and other institutions, aneusomy of chromo- of 8q has been reported to often accompany the allelic loss in 8p some 7 correlated with a less favorable phenotype (high tumor in prostate cancer (24) and hepatocellular cancer (25). Recent grade and advanced pathological stage; Refs. 7, 31, and 32). FISH studies of prostate cancers have confirmed that a loss of Also, gain of chromosome 7 (trisomy 7) has been reported as a signals at 8p22 often correlate with a gain of centromere 8 consistent anomaly in other solid tumors such as urinary blad- signals (26-28). A likely genetic mechanism underlying both der, brain, colon, and kidney (4, 33), suggesting a common locus the FISH and molecular genetic observations is the acquisition for carcinogenesis. We performed a preliminary case-control of multiple isochromosomes 8q in tumor cells. Interestingly, FISH study with pathological stage B and C patients who died amplification of the myc oncogene, which is located on 8q24, is from metastatic prostate cancer within 3 years after radical reported to be correlated with a shorter disease-free interval and prostatectomy. We found that aneusomy of chromosome 7 is a

A 100- 100 A B -- All (n = 181)

so- 80 ,.,-,_, (16)

so 60 (52) "~.., l o. (7) :__.] 1) ._o 40- o 40 E T= -- Nonaneuploid (n = 102) :...... ® ..... Aneuploid (n = 79) (1) 20- o~ 20- # P= 0.060 0 ~ 1.0 1'5 0 ~ 1.0 1'5 Years Years

-- No gain of chromosome 8 (n = 142) C 100- ..... Gain of chromosome 8 (n = 39) U 100 -- Apparently normal Y (n = 165) A ..... Aneusomy Y (n = 16)

.~_ 80- (11) .~ 80 L___: ~ (14) L, ii '11 65 ~"~...... I "= 80 (21 ) % I 0O) (31. ~"...... ~ [ (2) ~...... i .O 40 ~,1.; ,,, t .2 40 (2) l I (2) E (5) k .... E I i ® ~, 2o 20 o Z P =0.006 P=0.021 0 0 5' 10 1'5 ; 5 1.0 1'5 Years Years

Fig. 1 Systemic cancer progression-free rate after radical prostatectomy for the 181 patients with histological high-grade pathological stage C (pT3NoMo) prostate cancer. A, overall; B, according to FISH aneuploidy (log rank test; P = 0.060); C, according to gains of chromosome 8 (P = 0.006); and D, according to aneusomy of chromosome Y (P = 0.021).

(167) "¢Z~...... -::.~_~ (93) 100 -- All (n = 181) S 100- A A (23) 80 80- (64) ,.~..~ ......

E (11) ~'--i (2) 60 I 60 i. o o .=E_- Nonaneuploid (n = 102) L 4o 0. 4o ¢o ..... Aneuploid (n = 79) (2) ,L 20 O P=0.38 0 ; 1.0 1'5 o Years Years C 100- ~2~ Dloo~. 147) A

80" .... , ...... 80 :---: E.-I (lOl ,4.; ¢n 60- (6) l__.. ~ ~ 6o. o ) .~ '[ ...... F.... L(4) (2) Q. 40 (1) ~ 4o 00 -- No gain of chromosome 8 (n = 142) °m i Apparently normal Y (n = 165) ..... Gain of chromosome 8 (n = 39) Oo ..... Aneusomy Y (n = 16) °~ 20 2O 0 P=0.24 P<0.~I 0 ; 1.0 1'5 o ~ ; 1'o 1'6 Years Years

Fig. 2 Cancer-specific survival after radical prostatectomy for the 181 patients with histological high-grade pathological stage C (pT3NoM o) prostate cancer. A, overall; B, according to FISH aneuploidy (log rank test; P = 0.38); C, according to gains of chromosome 8 (P = 0.24); and D, according to aneusomy of chromosome Y (P < 0.001).

nonrandom alteration that correlates with clinically aggressive tion for deoxyribonucleic acid ploidy analysis of prostatic adenocarci- prostate cancer (8). For this current investigation, we used the noma. J. Urol., 150: 120-125, 1993. same FISH methodology and criteria for FISH aneusomy as in 6. Brown, J. A., Alcaraz, A., Takahashi, S., Persons, D. L., Lieber, M. M., and Jenkins, R. B. Chromosomal aneusomies detected by fluo- the preliminary case-control study. The reason for the discordant rescence in situ hybridization (FISH) in clinically localized prostate results regarding aneusomy of chromosome 7 is unclear. One carcinoma. J. Urol., 152: 1157-1162, 1994. possibility could be the difference of the patient groups studied. 7. Takahashi, S., Qian, J., Brown, J. A., Alcaraz, A., Bostwick, D. G., The previous case-control study included pT2NoM o tumors and Lieber, M. M., and Jenkins, R. B. Potential markers of prostate cancer used patients with a "very poor prognosis" (e.g., patient death aggressiveness detected by fluorescence in situ hybridization in needle within 3 years after surgery; Ref. 8). In addition, the definition biopsies. Cancer Res., 54: 3574-3579, 1994. 8. Alcaraz, A., Takahashi, S., Brown, J. A., Herath, J. F., Bergstralh, of gain and aneusomy used in our studies could be fulfilled by E. J., Larson-Keller, J. J., Lieber, M. M., and Jenkins, R. B. Aneuploidy a small subpopulation of tumor cells. Thus, it is possible that a and aneusomy of chromosome 7 detected by fluorescence in situ hy- biological character of the residual majority of tumor cells bridization are markers of poor prognosis in prostate cancer. Cancer influences the patient prognosis. The criteria for gain and aneu- Res., 54: 3998-4002, 1994. somy used were established based on the consistent results of 9. Utz, D. C., and Farrow, G. M. Pathologic differentiation and prog- our previous studies (6-8). We believe that these criteria are nosis of prostatic carcinoma. J. Am. Med. Assoc., 209: 1701-1703, 1969. reasonable for this study and that changing these criteria to 10. Zincke, H., Fleming, T. R., Furlow, W. L., Myers, R. P., and Utz, better stratify patients according to clinical outcome is statisti- D. C. Radical retropubic prostatectomy and pelvic lymphadenectomy cally inappropriate (34). Because of the consistent incidence of for high-stage cancer of the prostate. Cancer (Phila.), 47: 1901-1910, the aneusomy of chromosome 7 in prostate cancer (6-8, 32, 1981. 33), additional studies to evaluate the prognostic relevance of 11. Cheng, W. S., Frydenberg, M., Bergstralh, E. J., Larson-Keller, J. J., this common numeric alteration in prostate carcinoma will be and Zincke, H. Radical prostatectomy for pathologic stage C prostate important. cancer: influence of pathologic variables and adjuvant treatment on disease outcome. Urology, 42: 283-291, 1993. In this retrospective analysis, FISH signals were enumer- 12. Nativ, O., Winkler, H. Z., Raz, Y., Therneau, T. M., Farrow, G. M., ated by at least two independent individuals, and all investiga- Myers, R. P., Zincke, H., and Lieber, M. M. Stage C prostatic adeno- tions were carried out without knowledge of the clinicopatho- carcinoma: flow cytometric nuclear DNA ploidy analysis. Mayo Clin. logical characteristics and survival data of the patients studied. Proc., 64: 911-919, 1989. We believe that this study was performed with acceptable min- 13. Nativ, O., Myers, R. P., Farrow, G. M., Therneau, T. M., Zincke, H., imum bias, and that aneuploidy and gains and aneusomies of and Lieber, M. M. Nuclear deoxyribonucleic acid ploidy and serum chromosomes 8 and Y detected by FISH are new potential prostate specific antigen in operable prostatic adenocarcinoma. J. Urol., 144: 303-306, 1990. markers for a poor prognosis in pathological stage C prostate 14. Sandberg, A. A. Chromosome abnormalities and related events in carcinoma. These findings may be of important practical use, prostate cancer. Hum. Pathol., 23: 368-380, 1992. since we have reported a methodology to perform rapid FISH 15. Lundgren, R., Mandahl, N., Heim, S., Limon, J., Henrikson, H., and chromosome analysis on pretreatment prostate needle biopsy Mitelman, F. Cytogenetic analysis of 57 primary prostatic adenocarci- core specimens that can subsequently be used for routine his- nomas. Genes Chromosomes & Cancer, 4: 16-24, 1992. topathological examination (7). The pretreatment detection of 16. Heim, S. Cytogenetics in the investigation of hematological disor- ders. Baillieres Clin. Hematol., 3: 921-948, 1990. cytogenetic markers associated with systemic cancer progres- 17. Walter, T. A., Berger, C. S., and Sandberg, A. A. The cytogenetics sion and cancer death described above may prove useful for of renal tumors. Where do we stand, where do we go? Cancer Genet. selecting patients who would benefit from early aggressive Cytogenet., 43: 15-34, 1989. systemic adjuvant treatment, while minimizing the therapeutic 18. Bigner, S. H., Mark, J., and Bigner, D. D. Cytogenetics of human burden to those who do not require it. brain tumors. Cancer Genet. Cytogenet., 47: 141-154, 1990. 19. Kovacs, G., and Frisch, S. Clonal chromosome abnormalities in tumor cells from patients with sporadic renal cell carcinomas. Cancer REFERENCES Res., 49: 651-659, 1989. 1. Gittes, R. F. Carcinoma of prostate. N. Eng. J. Med., 324: 236-245, 20. Breitkreuz, T., Romanakis, K., Lutz, S., Seitz, G., Bonkhoff, H., 1991. Unteregger, G., Zwergel, T., Zang, K. D., and Wullich, B. Genotypic characterization of prostatic carcinomas: a combined cytogenetic flow 2. Shankey, V., Kallioniemi, O., Koslowski, J. M., Lieber, M. M., cytometry and in situ DNA hybridization study. Cancer Res., 53: Mayall, B. H., Miller, G., and Smith, G. J. Consensus review of the 4035-4040, 1993. clinical utility of DNA content cytometry in prostate cancer. Cytometry, 21. Baretton, G. B., Vallina, C., Vogt, T., Schneiderbanger, K., 14: 497-500, 1993. Diebold, J., and Lrhrs, U. Interphase cytogenetic analysis of prostatic 3. Devilee, P., Thierry, R. F., Kievits, T., Kolluri, R., Hopman, carcinomas by use of nonisotopic in situ hybridization. Cancer Res., 54: A. H. N., Willard, H. F., Pearson, P. L., and Cornelisse, C. J. Detection 4472-4480, 1994. of chromosome aneuploidy in interphase nuclei from human primary 22. Emi, M., Fujiwara, Y., Nakajima, T., Tshuchiya, E., Tsuda, H., breast tumors using chromosome-specific repetitive DNA probes. Can- Hirohashi, S., Maeda, Y., Tsuruta, K., Miyaki, M., and Nakamura, Y. cer Res., 48: 5825-5830, 1988. Frequent loss of heterozygosity for loci on chromosome 8p in hepato- 4. Waldman, F. M., Carroll, P. R., Kerschmann, R., Cohen, M. B., cellular carcinoma, colorectal cancer, and lung cancer. Cancer Res., 52: Field, F. G., and Mayall, B. H. Centromeric copy number of chromo- 5368-5372, 1992. some 7 is strongly correlated with tumor grade and labeling index in 23. Fujiwara, Y., Emi, M., Ohata, H., Kato, Y., Nakajima, T., Moil, T., human bladder cancer. Cancer Res., 51: 3807-3813, 1991. and Nakamura, Y., Evidence for the presence of two tumor suppressor 5. Persons, D. L., Gibney, D. J., Katzmann, J. A., Lieber, M. M., genes on chromosome 8p for colorectal carcinoma. Cancer Res., 53: Farrow, G. M., and Jenkins, R. B. Use of fluorescent in situ hybridiza- 1172-1174, 1993.

24. Bova, G. S., Carter, B. S., Bussemakers, M. J. G., Emi, M., 30. Van Den Berg, C., Guan, X., Von Hoff, D., Jenkins, R. B., Bittner, Fujiwara, Y., Krprianou, N., Jacobs, S. C., Robinson, J. C., Epstein, J. I., M., Griffin, C., Kallioniemi, O., Visakorpi, T., McGill, J., Herath, J. F., Walsh, P. C., and Isaacs, W. B. Homozygous deletion and frequent Epstein, J., Sorosdy, M., Meltzer, P., and Trent, J. DNA sequence allelic loss of chromosome 8p22 loci in human prostate cancer. Cancer amplification in human prostate cancer identified by chromosome mi- Res., 53: 3869-3873, 1993. crodissection: potential prognostic implications. Clin. Cancer Res., 1: 25. Fujiwara, Y., Monden, M., Mori, T., Nakamura, Y., and Emi, M. 11-18, 1995. Frequent multiplication of the long arm of chromosome 8 in hepatocel- 31. Bandyk, M. G., Zhao, L., Troncoso, P., Pisters, L. L., Palmer, J. L., lular carcinoma. Cancer Res., 53: 857-860, 1993. von Eschenbach, A. C., Chung, L. W. K., and Liang, J. C. Trisomy 7: 26. Macoska, J. A., Micale, M. A., Sakr, W. A., Benson, P. D., and a potential cytogenetic marker of human prostate cancer progression. Wolman, S. R. Extensive genetic alterations in prostate cancer revealed Genes Chromosomes & Cancer, 9: 19-27, 1994. by dual PCR and FISH analysis. Genes Chromosomes & Cancer, 8: 88-97, 1993. 32. Zitzelsberger, H., Sztics, S., Weier, H. U., Lehmann, L., Brasel- mann, H., Enders, S., Schilling, A., Breul, J., H6fler, H., and Bauch- 27. Macoska, J. A., Trybus, T. M., Sakr, W. A., Wolf, M. C., Benson, inger, M. Numerical abnormalities of chromosome 7 in human prostate P. D., Powell, I. J., and Pontes, J. E. Fluorescence in situ hybridization cancer detected by fluorescence in situ hybridization (FISH) on paraffin- analysis of 8p allelic loss and chromosome 8 instability in human embedded tissue sections with centromere-specific DNA probes. J. prostate cancer. Cancer Res., 54: 3824-3830, 1994. Pathol., 172: 325-335, 1994. 28. Matsuyama H., Pan, Y., Skoog, L., Tribukait, B., Naito, K., Ekman, P., Lichter, P., and Bergerheim, U. S. Deletion mapping of chromosome 33. Johansson, B., Heim, S., Mandahl, N., Mertens, F., and Mitelman, 8p in prostate cancer by fluorescence in situ hybridization. Oncogene, 9: F. Trisomy 7 in nonneoplastic cells. Genes Chromosomes & Cancer, 6: 3071-3076, 1994. 199-205, 1993. 29. Borg, ,~., Baldetorp, B., Fern6, M., Olsson, H., and Sigurdsson, H. 34. Altman, D. G., Lausen, B., Sauerbrei, W., and Schumacher, M. c-myc amplification is an independent prognostic factor in postmeno- Dangers of using "optimal" cutpoints in the evaluation of prognostic pausal breast cancer. Int. J. Cancer, 51: 687-691, 1992. factors. J. Natl. Cancer Inst., 86: 829-835, 1994.

Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 1996 American Association for Cancer Research. Aneusomies of chromosomes 8 and Y detected by fluorescence in situ hybridization are prognostic markers for pathological stage C (pt3N0M0) prostate carcinoma.

S Takahashi, A Alcaraz, J A Brown, et al.

Clin Cancer Res 1996;2:137-145.

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