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The prognostic value of Ki-67, p53, epidermal growth factor receptor, 1p36, 9p21, 10q23, and 17p13 in skull base chordomas.

Chordomas are rare neoplasms that may arise from persistent notochord tissue. These tumors can arise anywhere along the axial spine but most commonly are seen in the skull base/clivus and sacrococcygeal regions. Three variants of chordoma are recognized histologically: classic, chondroid, and dedifferentiated/sarcomatoid. Although typically slow growing, chordomas are extremely difficult to eradicate by surgical and adjuvant means. Thus, they usually recur multiple times, causing considerable mortality as evidenced by 5- and 10-year survival rates between 70% and 80% and 30% and 40%, respectively. (1) Because growth rate, invasiveness, and survival after initial resection is highly variable, identification of molecular and protein prognostic markers is of interest.

In broad-based genetic studies, most chordomas were found to be diploid, with reduced survival in cases that had abnormal karyotypes including aneuploidy. (2,3) Additional work has identified nonrandom patterns of copy number variation, including losses on 1p, 3, 9p, and 10, as well as gains on 7, with no site-specific differences. (4-9) In particular, the CDKN2A locus encoding the tumor suppressor protein p16 has been found to be frequently deleted. (5) Several familial chordoma cohorts have been studied extensively, with genetic mapping identifying 1p36 and 7q as key sites of loss and gain, respectively. (10-13) 1p36 loss is also seen in many sporadic chordomas (14) and was suggested by 1 group to be an unfavorable prognostic marker. (15)

Expression of receptor tyrosine kinases (RTKs) and cell cycle proteins has also been studied in chordomas, with conflicting results. Some have shown a correlation between increased Ki-67/MIB-1 proliferation index (PI) and solid pattern, recurrent tumors, shorter disease-free survival, overall survival, and/or doubling time but not others. (16-21) Aberrant overexpression of p53 correlates with increased PI and/or shorter survival in some studies, while others have not found any such link. (19-23) Epidermal growth factor receptor (EGFR), an RTK well known to have oncogenic activity in a variety of neoplasms, has been consistently shown to be overexpressed and activated in chordomas, often in conjunction with other RTKs including platelet-derived growth factor receptors A and B, c-KIT, and HER2/neu. (24-26) One such study found no association with RTK expression and tumor site or survival,25 but another showed that absence of c-MET correlated with an adverse outcome. (27)

Thus, although chordomas are known to carry nonrandom genetic alterations and variable expression of proteins involved in cellular proliferation, a consensus regarding the prognostic utility of these biomarkers has not yet been reached. Herein we describe our retrospective study of primary clival chordoma specimens resected from 28 patients. Fluorescence in situ hybridization (FISH) on 1p36 and 9p21, and polymerase chain reaction (PCR)-based microsatellite loss of heterozygosity (LOH) analysis of 1p, 9p, 10q, and 17p, as well as immunohistochemical evaluation of Ki-67 PI, p53, and EGFR, was performed. Data were then correlated with clinical outcomes. These results suggest that either loss at 9p21 or Ki-67 PI more than 5% are adverse prognostic markers. In particular, to our knowledge this study is the first to demonstrate that 9p21 deletion is an adverse prognostic biomarker. Incorporation of these markers into routine evaluation of chordomas may thus help customize postsurgical monitoring and therapeutic stratification.

MATERIALS AND METHODS

Cohort Characteristics

The chordoma cohort used in this study was a subset of cases from a larger cohort described previously. (28) All tumors were initial resections; recurrences and previously treated tumors were excluded. Paraffin blocks and slides from 28 patients were successfully retrieved from the University of Pittsburgh Medical Center, Department of Pathology Archives (1969-2007) in accordance with institutional review board guidelines. The diagnosis of chordoma and histologic subtyping on hematoxylin-eosin stains were verified by 2 observers. Clinical follow-up data were available for all but 1 patient. The precise cause of death (ie, death from tumor or other disease) was not available in most cases. Median follow-up interval on surviving patients was 67 months (range, <1-212 months). Cohort characteristics are summarized in Table 1. Of note, not every FISH and immunohistochemical study was successful in all 28 cases; hence the denominators lower than 28 in "Results."

Tissue Microarray Construction and Immunohistochemistry

The method for tissue microarray construction was described previously. (28) Briefly, a manual tissue arrayer (MTA-1, Beecher Instruments, Sun Prairie, Wisconsin) was used to select 0.6-mm cores from each paraffin block to a blank recipient paraffin block and arrayed in triplicate. Use of decalcified blocks could not be avoided in 50% (13 of 26) of the cases. Paraffin sections from the constructed tissue microarray block were cut and incubated with antibodies to p53 (1:100; DO7, Dako, Carpinteria, California), Ki67 (1:25; ki-55, Dako), and EGFR (1:500; H11, Dako). Staining was visualized using the ImmPRESS (Vector Labs, Burlingame, California) detection system with 2-diaminobenzidine as the substrate chromogen. Each antibody was scored manually between 2 observers. Only cases with strong nuclear staining in most cells were considered positive. Ki-67 PI was determined for each core and assigned a score of 0 if less than 5% or 1 if greater than or equal to 5%. Cells with any EGFR staining of both cytoplasm and membrane were considered positive. Discrepancies between observers were resolved by simultaneous review.

Fluorescence In Situ Hybridization

Fluorescence in situ hybridization protocols were similar to that described previously. (29) Formalin-fixed paraffin-embedded tissue microarray sections were mounted and serially sectioned at 5-|im intervals. Hematoxylin-eosin sections were used to determine the area of the tissue to be targeted for analysis. Fluorescence in situ hybridization slides were deparaffinized in xylene twice for 10 minutes, dehydrated twice with 100% ethanol, and then pretreated using the Vysis Paraffin Pretreatment Kit (Abbot Molecular Inc, Des Plaines, Illinois). Slides were digested for 18 minutes in protease solution (0.5 mg/mL) at 37[degrees]C. For 1p36, hybridization was performed using a spectrum orange labeled probe for 1p36 and spectrum green labeled control probe for 1q25 (Vysis dual-color probe set, LSI 1p36/LSI 1p25, Vysis, Downers Grove, Illinois). For 9p21, hybridization was performed using a spectrum orange labeled probe for CDKN2A (p16) and a spectrum green labeled chromosome 9 centromeric probe (CEP 9) (Vysis). The target slide and probe were codenatured at 95[degrees]Cfor8minutes and incubated overnight at 37[degrees]C in a humidified chamber. Posthybridization washes were performed using 2XSSC/0.3% Igepal at 72[degrees]C for 2 minutes. Slides were air dried in the dark and counterstained with DAPI (Vysis). Analysis was performed using a Nikon Optiphot-2 (Nikon Inc, Melville, New York) and Quips Genetic Workstation equipped with Chroma Technology filter set with single band excitors for Spectrum-Orange, Fluorescein isothiocyanate, 4'6-diamidino-2-pheyndole, (ultraviolet 360 nm) (Vysis). Only individual and well-delineated cells were scored. Overlapping cells were excluded from the analysis. At least 60 nuclei were counted in each case. Cases were considered positive for 1p36 deletion if 20% or more of nuclei showed deletion. For 9p21, only homozygous deletion was counted and was defined by loss of both 9p21 signals in 20% or more of nuclei with at least one CEP 9 signal. These cut-off points were derived using nonneoplastic autopsy brain tissue as controls.

PCR-Based Microsatellite LOH Analysis

Manual microdissection of the tissue sample was performed to include tumor tissue. Matched nonneoplastic tissue was not available. Specimens with the minimum of 50% of tumor cells in a microdissection target were accepted for the analysis. DNA was isolated using standard laboratory procedures. Optical density readings were obtained. The assay used 7 microsatellite markers on chromosome 1p22-36 (D1S171, D1S162, D1S199, D1S1172, D1S1161, D1S407, and D1S226), 3 on 9p21-22 (CDKN2A gene), 3 on 17p13 (TP53 gene), and 2 on 10q23 (PTEN gene). Polymerase chain reaction was performed and the PCR products were analyzed using capillary gel electrophoresis on GeneMapper ABI 3730 (Applied Biosystems, Foster City, California). Relative fluorescence was determined for individual alleles and the ratio of peaks was calculated. Neoplastic tissue was then analyzed to detect LOH. Because normal tissue was not available, peak height ratios falling outside of 2 standard deviations beyond the mean of previously validated normal values for each polymorphic allele paring were assessed as showing LOH. Only LOH at 2 or more informative loci on 1p and 9p, and LOH at 1 or more informative loci on 10q or 17p, were scored as loss.

Data Analysis

Survival analysis was performed using the Kaplan-Meier method with SPSS software, version 17.0 (SPSS, Chicago, Illinois). Group-wise comparisons were made using the log-rank test.

RESULTS

Cohort Demographics and Histology

Our retrospective cohort consisted of 28 clival chordomas resected from 11 female and 17 male patients, with a median age of 39 years (range, 12-77 years) (Table 1). At the end of the study, 10 patients had died and 1 was lost to follow-up with a (Kaplan-Meier) median survival of 169 months. The (arithmetic) median follow-up on surviving patients was 67 months. Thiry-nine percent (11 of 28) of the chordomas were conventional (Figure 1, A and B), 61% (17 of 28) were chondroid (Figure 1, C), and none were dedifferentiated. Thirty-two percent (9 of 28) had a solid component greater than 5% (Figure 1, B).

[FIGURE 1 OMITTED]

Ki-67, p53, and EGFR Immunohistochemistry

Immunohistochemical analysis of Ki-67 showed that 32% (8 of 25) of the tumors had nuclear Ki-67 expression in 5% or more of tumor cells (Table 2; Figure 1, D). Such cases had a 56% reduction in mean survival time compared with patients whose chordomas had a PI less than 5% (69.3 versus 159.3 months, P = .005) (Figure 3, A).

p53 accumulation was seen in 44% (11 of 25) of tumors (Figure 1, E). Survival in patients with tumors expressing low levels of p53 (145.1 months, confidence interval [CI] = 102.3-187.9) was not significantly different from those with increased p53 expression (96.6 months, CI = 65.4127.8, P = .28).

Epidermal growth factor receptor expression was uncommon in chordomas, with only 8% (2 of 24) of cases showing any positivity (Figure 1, F). Although survival in these 2 patients was shorter than those with EGFR-negative tumors, the difference was not statistically significant (57.7 months, CI = 16.4-98.9 versus 137.6 months, 99.1-176.1, P = .27).

FISH and PCR-Based LOH on 1p, 9p, 10q, and 17p

Large LOH of 1p was seen in 38% (9 of 24) of tumors (Figure 2, A and C), while 17% of chordomas had hemizygous loss of the 1p36 locus (4 of 23; Figure 2, E and G; Table 2). Loss of heterozygosity on 1p (120.4 months, CI = 87.5-153.2) was not associated with a difference in survival compared with 1p intact tumors (142.0 months, CI = 91.0-192.9, P = .82) (Figure 3, D). Likewise, overall survival was similar in 1p36 deleted tumors (122.7 months, CI = 91.7-153.6) versus 1p36 intact tumors (134.5 months, CI = 97.2-171.7, P = .63).

Microsatellite-based LOH analysis identified 9p loss in 12% of tumors (Figure 2, B and D), while FISH identified homozygous loss of the 9p21 locus in 22% (5 of 23) of cases (Figure 2, F and H; Table 2). 9p loss via LOH analysis was associated with a 51% decrease in mean survival time (71.7 months, CI 42.5-100.9) compared with 9p intact cases (146.2 months, CI 109.8-182.5, P = .03) (Figure 3, B). Survival in tumors with 9p21 homozygous deletion showed a similar trend toward reduced survival compared with 9p21 intact cases (73.8 months, CI 47.9-99.7 versus 151.4 months, CI 115.1-187.7, P = .08) (Figure 3, C). Thirty-five percent (9 of 26) of tumors had chromosome 9 monosomy in at least some cells but did not correlate with survival (not shown).

Of note, correlation between FISH and PCR-based LOH results on 1p and 9p was not perfect. Two of 4 cases with 1p36 loss on FISH showed LOH of 1p, whereas 6 of 8 cases with 1p LOH did not show any deletion of 1p36 (LOH succeeded but FISH failed on the ninth case). Likewise, 3 of 5 cases with homozygous 9p21 deletion via FISH did not show LOH on 9p, while 1 of 3 tumors had 9p LOH but not 9p21 homozygous deletion.

Loss of heterozygosity of 10q23 and 17p13 showed no correlation with overall survival (Table 2; Figure 3, E and F), and 17p LOH did not correlate with increased p53 staining (not shown).

COMMENT

The primary therapy in chordoma management is surgical resection, although the recurrence rate is so high that it is generally regarded as incurable even with radical resection. (30) High-dose radiotherapy has demonstrated some success in delaying recurrence and progression, but because the tumor is usually located in a delicate area and shows a wide range of invasiveness and tumor doubling time, oncologists have difficulty deciding whether postsurgical radiation is always appropriate on a case-by-case basis, or how often to monitor patients for recurrence. Thus, effective biomarkers that predict behavior would be most useful in the management of these tumors. Our results in a retrospective cohort of clival chordomas suggest that loss of 9p21 or Ki-67 PI more than 5% are adverse prognostic markers associated with reduced survival. In particular, detection of LOH in at least 2 microsatellite loci on 9p21 was significantly associated with shorter survival, while homozygous deletion of 9p21 approached significance. p53 and EGFR expression and loss of 1p36,10q, or 17p, on the other hand, did not correlate with survival in this analysis.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Ki-67 is a protein expressed in cells that are not in the [G.sub.0] phase of the cell cycle and appears to play a role in chromatin structuring during replication. (31) It has been used for decades as a marker of cellular proliferation in both neoplastic and nonneoplastic conditions and often correlates with biologic behavior in a variety of tumors. In other studies on chordomas, the average PI was usually between 2% and 5%, in keeping with its overall slow growth rate. Our results are consistent with most prior work, wherein chordomas with a PI more than 5% had a shorter doubling time and/or shorter overall survival, (16,18-20) but not all studies have found such a link. (17,21)

Loss of 1p and/or 1p36 was identified by both FISH and LOH in nearly 40% of chordomas. These results are consistent with prior work showing that such losses are quite common in chordomas, with reported frequencies varying between 30% and 85%. (4,14,15) 1p LOH or 1p36 deletion did not correlate with survival in this cohort, in contrast to the only other study wherein 1p36 status and outcome was analyzed. (15) An isochromosome 1q has been reported in a subset of recurrent chordomas, (6) and familial kindreds of clival chordomas often feature deletion of 1p36 as a key molecular defect. (10) Together, these findings reinforce the idea that key tumor suppressor genes may be located on 1p, in particular 1p36, but the precise genes involved are unclear. Although this locus is likely critical in oncogenesis, its utility as a prognostic biomarker is questionable.

In this study, 9p losses were less common than 1p losses; nearly 25% of all chordomas had 9p loss detectable by either FISH or LOH assays. Specific deletions of CDKN2A on 9p21 have been identified in up to 70% of all chordomas, and 9p21 has been shown to be lost in unbalanced translocations with 1p36 in familial tumors. (5,7,10) The current study, however, is the first to correlate 9p21 loss with worse outcome. That such a deletion could promote aggressive behavior is readily explained by the presence of the aforementioned CDKN2A gene on 9p21, which encodes the critical cell cycle checkpoint protein p16. Homozygous loss of this gene and/or loss of p16 expression are well-known markers of high-grade behavior in a variety of neoplasms, including gliomas. (32) Other genes on 9p21 could be involved, but as is the case with the 1p36 locus such candidates are speculative as of yet.

The relatively low correlation between FISH and LOH analyses of 1p and 9p loci is interesting, and such discrepancies have been documented previously. (33) There are several possible reasons for this. One, the FISH probes for 1p36 and 9p21 are commercial, proprietary probes for which the exact gene sequence is not publicly available. So other than knowing that the probes are designed to target 1p36 and include the CDKN2A region of 9p21, respectively, exact correlation with specific microsatellite loci is not possible. Small losses could thus be detected by one assay and not the other if the FISH probes do not include the microsatellite sequences. In particular, the commercially available 9p21 LSI probe is rather large (190 kb) and also covers p14, p15, and a portion of the MTAP gene that is frequently codeleted with the CDKN2Ap16 gene. In our study we did not investigate status of these genes by LOH analysis, but we believe that deletion of these genes might be an underlying reason for deletions detected by FISH but not PCR-based LOH analysis. Another possibility is that because matched nonneoplastic DNA was not available, the increased frequency of noninformative loci may have obscured true LOH in some cases, particularly those that had homozygous deletion at similar loci by FISH. Third, this cohort is relatively small due to the rarity of chordomas. In a larger cohort, stronger correlation between FISH and LOH might be found.

Loss on 10q23 has drawn interest in oncology research because a key gene, PTEN, encoding the phosphatase and tensin homolog protein, is located on 10q23 and is mutated or deleted in many neoplasms. Loss of PTEN functioning results in unregulated PI3K/Akt/mTOR pathway activation, promoting cellular proliferation, migration, and radioresistance. (34) It may also act to promote overall genomic stability. Loss of 10q has been identified as a high-grade marker and/or an adverse prognostic indicator in many tumors including gliomas. In chordomas, either part of the long arm or all of chromosome 10 has been shown to be deleted in up to 70% of cases, including 1 kindred of familial tumors. (5,7,9,10) In this series, 10q23 LOH was seen in 57% of all tumors with informative loci but did not correlate with a higher PI or shorter survival. Nevertheless, the frequency of 10q23 deletion suggests that this region contains key genes in tumorigenesis, including PTEN.

TP53 is a well-studied tumor suppressor gene located on 17p13. It is such a critical regulator of the cell cycle that more than half of all human neoplasias contain some alteration of the protein. A commonly used pair of surrogate markers for identifying a p53-driven tumor when a full mutation analysis is not possible is 17p13 LOH and accumulation of p53 via immunohistochemistry. It should be noted, though, that mechanisms of p53 inactivation and mutations are complex and incompletely understood, and there are most likely other important tumor suppressors on 17p13. TP53 mutations have yet to be detected in chordomas by sequencing, (23) but accumulation of p53 has been described in multiple studies with about 30% of cases showing at least some immunopositivity, a proportion similar to this cohort, in agreement with some studies17,20 but not others. (19,23)

Prior work has identified EGFR and other RTK activity in some chordomas. (26) Only one study has identified a link between RTK expression and survival in chordomas, wherein presence of c-MET expression was associated with favorable outcome. (27) In our cohort, all cases showed strong c-MET expression (data not shown), rendering evaluation of its prognostic utility impossible. Nevertheless, the suggestion that chordomas do express RTKs raises the possibility of targeted therapy with a small molecule inhibitor like erlotinib or imatinib.35 It will be of interest to determine whether RTK status can predict response to such inhibitors, as is routinely done with trastuzumab in breast carcinomas.

In summary, our findings suggest that biomarkers enhancing prognostic accuracy in chordomas exist. 9p21/ CDKN2A deletion is associated with shorter survival, a novel finding in chordomas. This study also supports prior results suggesting that Ki-67 PI is a simple, costeffective method to identify tumors at increased risk of aggressive behavior. To confirm these findings, an institutional prospective study with more complete clinical characterization is currently underway. Chordomas may therefore soon join the growing list of tumors for which prognostic and predictive tests routinely enhance quality of care.

We thank Kimberly Fuhrer, AS, and Cary Sipos, BS, for their histologic and immunohistochemical expertise, as well as John Salvatore and Carol Sherer, MS, for their work in fluorescence in situ hybridization. We would also like to thank Jennifer Ridge Hetrick, BS, RHIA, CTR, and Kim Adams, BS, for their

administrative support. This work was supported by a Callie Rohr/American Brain Tumor Association Fellowship (Dr Horbinski), a pilot study from the Head and Neck SPORE NIH 1P50 CA097190 (awarded to Dr Seethala), the Stout Family Fund for Head and Neck Cancer Research at the Eye & Ear Foundation of Pittsburgh, and the Head and Neck Oncology Registry.

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Craig Horbinski, MD, PhD; Gerard J. Oakley, MD; Kathleen Cieply, MS; Geeta S. Mantha, PhD; Marina N. Nikiforova, MD; Sanja Dacic, MD, PhD; Raja R. Seethala, MD

Accepted for publication December 4, 2009.

From the Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania.

The authors have no relevant financial interest in the products or companies described in this article.

Presented in part at the annual meeting of the United States and Canadian Academy of Pathology, Boston, Massachusetts, March 9, 2009.

Reprints: Raja Seethala, MD, Presbyterian University Hospital, Department of Pathology, A616.3, 200 Lothrop St, Pittsburgh, PA 15213 (e-mail: seethalarr@upmc.edu).
Table 1. Chordoma Cohort Demographics and
Histologic Featuresa

Demographics Histology

Median age, 39 y (range, 12-77 y) Conventional, 39% (11/28)
61% male Chondroid, 61% (17/28)
39% female >5% solid, 32% (9/28)
Median follow-up time, 67 mo
36% dead

(a) This cohort reflected the age and gender distribution of skull
base chordomas. Most cases showed at least partial chondroid
differentiation, while 32% showed a significant solid component.

Table 2. Correlation of Immunohistochemical and Molecular Biomarkers
With Survival in Skull Base Chordomas (a)

 Frequency Mean OS,
Biomarker Result (%) mo

Ki-67 PI <5% 17/25 (68) 159.3
 [greater than 8/25 (32) 69.3
 or equal to] 5%
p53 expression <25 14/25 (56) 145.1
 score [greater than 11/25 (44) 96.6
 or equal to] 25
EGFR expression Negative 22/24 (92) 137.6
 Positive 2/24 (8) 57.7
1p36 FISH Intact 19/23 (83) 134.5
 Hemizygous deletion 4/23 (17) 122.7
1p LOH Negative 15/24 (62) 142.0
 Positive 9/24 (38) 120.4
9p21 FISH Intact 18/23 (78) 151.4
 Homozygous deletion 5/23 (22) 73.8
9p LOH Negative 22/25 (88) 146.2
 Positive 3/25 (12) 71.7
10q LOH Negative 9/21 (43) 130.7
 Positive 12/21 (57) 117.1
17p LOH Negative 11/23 (48) 120.4
 Positive 12/23 (52) 125.1

Biomarker Result 95% CI, mo P

Ki-67 PI <5% 118.9-199.7 .005
 [greater than 28.7-110.0
 or equal to] 5%
p53 expression <25 102.3-187.9 .28
 score [greater than 65.4-127.8
 or equal to] 25
EGFR expression Negative 99.1-176.1 .27
 Positive 16.4-98.9
1p36 FISH Intact 97.2-171.7 .63
 Hemizygous deletion 91.7-153.6
1p LOH Negative 91.0-192.9 .82
 Positive 87.5-153.2
9p21 FISH Intact 115.1-187.7 .08
 Homozygous deletion 47.9-99.7
9p LOH Negative 109.8-182.5 .03
 Positive 42.5-100.9
10q LOH Negative 75.8-185.6 .86
 Positive 83.3-150.9
17p LOH Negative 69.2-171.6 .57
 Positive 92.9-157.3

Abbreviations: CI, confidence interval; EGFR, epidermal growth factor
receptor; FISH, fluorescence in situ hybridization; LOH, loss of
heterozygosity; OS, overall survival; PI, proliferation index.

(a) Of the biomarkers studied, Ki-67 proliferation index [greater than
or equal to] 5% and 9p LOH were significantly associated with shorter
OS, while chordomas with 9p21 homozygous deletion trended toward
shortened OS. p53 accumulation, EGFR expression, and deletions on 1p,
10q, and 17p were not significantly associated with survival.
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Author:Horbinski, Craig; Oakley, Gerard J.; Cieply, Kathleen; Mantha, Geeta S.; Nikiforova, Marina N.; Daci
Publication:Archives of Pathology & Laboratory Medicine
Date:Aug 1, 2010
Words:5061
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