Specific MicroRNAs Differentiate Adrenocortical Adenomas from Carcinomas and Correlate With Weiss Histopathologic System
Meora Feinmesser, MD,* Carlos Benbassat, MD,; Eti Meiri, PhD,¿ Hila Benjamin, MSc,¿ Danit Lebanony, MSc,¿ Yael Lebenthal, MD,§ Liat de Vries, MD,§ Tamara Drozd, MD,* and Yael Spector, MSc±
Abstract: MicroRNAs (miRs) play a central role in regulating gene expression and are strongly associated with cancer devel- opment. This study sought to determine if adrenocortical car- cinomas can be differentiated from adenomas by their miR profiles and to correlate the findings with the histologic Weiss system for identifying malignancy in adrenocortical tumors (ACTs). Forty-six primary and 2 recurrent ACTs retrieved from the files of the pathology department of a tertiary medical center were evaluated blindly for the Weiss criteria. High-quality RNA was extracted, and miR expression was evaluated with micro- arrays and quantitative reverse-transcriptase polymerase chain reaction. The Weiss system defined 17 tumors as carcinomas and 29 as adenomas. On microarray analysis, over a dozen miRs were upregulated or downregulated in carcinomas compared with adenomas. Upregulation of miR-503 was the best single discriminator of malignancy. The combination of miR-34a and miR-497 underexpression discriminated carcinomas from ad- enomas with 100% sensitivity and 96% specificity. Statistical analysis revealed a high level of correspondence between the Weiss system and miR expression. In conclusion, miR ex- pression can accurately identify malignant ACTs with equal efficiency to the Weiss system. miR analysis may have added value in tumors with borderline features that are difficult to interpret histopathologically.
Key Words: microRNAs, adrenocortical tumors, Weiss system (Appl Immunohistochem Mol Morphol 2015;23:522-531)
Received for publication February 22, 2014; accepted May 19, 2014.
From the Departments of *Pathology; Endocrinology, Beilinson Hospital, Rabin Medical Center (affiliated with Sackler School of Medicine, Tel Aviv University, Tel Aviv); §Department of Pediatric Endocrinology, Schneider Children’s Medical Center of Israel (affiliated with Sackler School of Medicine, Tel Aviv University, Tel Aviv), Petach Tikva; and įRosetta Genomics, Rehovot, Israel.
E.M., H.B., D.L., and Y.S. are full-time employees of Rosetta Genomics, which stands to gain from the publication of this manu- script. Authors M.F., and T.D., from clinical centers have received research funding from the company as part of this and other col- laborative projects.
Reprints: Meora Feinmesser, MD, Department of Pathology, Beilinson Hospital, Rabin Medical Center (affiliated with Sackler School of Medicine, Tel Aviv University, Tel Aviv), Petach Tikva 49100, Israel (e-mail: meoraf@clalit.org.il).
Copyright @ 2014 Wolters Kluwer Health, Inc. All rights reserved.
T he distinction of adrenocortical adenomas (ACAs) from adrenocortical carcinomas (ACCs) can pose a diagnostic challenge. To predict the malignant potential of adrenocortical tumors (ACTs), researchers have sug- gested methods of macroscopic assessment of tumor weight and size1-3 and several histopathologic scoring systems.49 Of these, the Weiss system is the most widely used and has been found to be the most reproducible.5,7-9 It is based on 9 microscopic features, most of which are typically related to malignancy and a few, such as the presence of eosinophilic cytoplasm in tumor cells, are specific to ACT morphology. The presence of ≥ 3 criteria in a given tumor indicates malignant potential, favoring the diagnosis of ACC; tumors with 2 features or less are more likely to be ACAs.7 However, some of the features show a poor correlation among pathologists, and some tumors present with borderline features that are difficult to interpret accurately.10 Oncocytic ACTs are especially problematic because they typically present with 3 Weiss criteria: predominantly eosinophilic cytoplasm, diffuse growth pattern, and pleomorphic nuclei.9,11-13 Therefore, numerous studies have proposed the use of im- munohistochemical and molecular biology techniques. Common immunohistochemical stains that may assist in the differentiation of ACCs from ACAs are vimentin and low-molecular-weight keratin: ACAs are more com- monly negative for vimentin and positive for low- molecular-weight keratin than ACCs.14,15 In 1 study, 74% of ACAs were positive on immunohistochemical staining for matrix metalloproteinase-2 compared with only 2% of adenomas.16 In a DNA microarray analysis, Giordano et al17 reported a several-fold differential ex- pression of 15 genes between ACCs and ACAs or normal adrenal tissue; the gene coding insulin-like growth factor was identified in > 80% of ACCs. Furthermore, aneu- ploidy was identified in 70% of ACCs and 20% of ACAs18,19; overexpression of p53 was found to be more common in carcinomas than adenomas20,21; and the clinical outcome of ACCs was found to be related to the genes DLG1 and PINK1.22 Overall information on the molecular pathogenesis of these tumors has been accu- mulating in recent years, but it is not routinely applied in the diagnostic setting.
MicroRNAs (miRs) are a family of short, noncoding RNAs with marked tissue specificity.23-25 They play a cen- tral role in the regulation of gene expression by binding to target mRNAs and are strongly associated with tissue and cancer development.25 Some miRs act as tumor-suppressor oncogenes26,27 by degrading specific mRNAs. Recent studies have described the deregulation of miRs in ACTs. 10,28-31 Soon et al,30 in a study of 22 ACCs and 27 ACAs diagnosed according to the Weiss system found 23 miRs that were significantly differentially expressed in ACC compared with ACA: 14 were upregulated and 9 downregulated. Most sig- nificantly, miR-335 and miR-195 were downregulated in ACCs. The combination of downregulation of miR-195 and upregulation of miR-483-5p in ACCs was associated with a detrimental outcome. Patterson et al29 examined 10 ACCs and 26 ACAs diagnosed according to clinical behavior at 2 years after diagnosis. ACCs showed downregulation of miR- 195 and upregulation of mir-483-5p, in addition to down- regulation of miR-100 and miR-125b. The ACTs could be categorized as benign or malignant by the expression of miR-483-5p. In a study of 36 samples of normal adrenal tissue and hormonally active and inactive ACAs and ACCs, Tömböl et al31 identified miRs with different expression in the ACCs: miR-184 and miR-503 were significantly upre- gulated, and miR-511 and miR-214 were significantly downregulated. The authors performed simultaneous miR and mRNA profiling in some of the cases to identify miR target molecules. Özata et al10 used a microarray platform to examine miR expression in 22 ACCs and 26 ACAs diag- nosed in accordance with the World Health Organization classification, as well as in the ACC cell line NCI-H25R. Similar to the previous studies, miR-483-3p, miR-483-5p, miR-210, and miR-21 were overexpressed in ACCs, and miR-195, miR-497, and miR-1974 were underexpressed. These findings were verified with a quantitative reverse- transcriptase polymerase chain reaction (qRT-PCR) plat- form in 25 ACCs and 43 ACAs.
Prompted by the data on miR expression in a broad spectrum of neoplastic processes, in addition to the data specific to ACTs, we sought to determine if differences in miR expression profile could distinguish ACCs from ACAs. We also examined correlations between the spe- cific miR profile of ACTs and the traditional Weiss sys- tem of histopathologic analysis.
MATERIALS AND METHODS
Sample Selection and Histopathologic Evaluation
Primary ACTs from 51 patients for which both clinical and macroscopic (weight and diameter) charac- teristics were available were retrieved from the files of the Department of Pathology of Rabin Medical Center, a tertiary university-affiliated hospital. The tumors were initially examined by a pathologist (T.D.) who selected the block for miR analysis. They were then reexamined by a second pathologist (M.F.) blinded to the original di- agnosis and clinical details who checked for the 9 histo- logic criteria of Weiss. Tumors that met ≥3 of the
criteria were considered malignant. For the molecular analysis, we used 52 tissue samples from the 51 affected patients, in addition to 2 recurrent ACTs that were ob- viously malignant (total, 54 samples from 53 patients) but for which the primary tumors were unavailable. Institu- tional review board approvals were obtained in accord- ance with the institute’s guidelines. Several samples did not contain sufficient tissue for analysis and were ex- cluded. The final analysis was conducted on 46 samples of primary tumors and 2 samples of recurrent tumors.
RNA Extraction
High-quality RNA was extracted from formalin-fixed paraffin-embedded samples containing over 50% tumor cellular content, as previously described.25 Briefly, the samples were deparaffinized with xylene, washed in ethanol, and digested with Proteinase K. RNA was extracted using acid-phenol:chloroform followed by ethanol precipitation and DNase digestion. Following a second acid-phenol: chloroform extraction, the pellet was resuspended in nu- clease-free water and analyzed for concentration and purity by spectrophotometry (NanoDrop1000; Thermo Fischer, Wilmington, DE).
Microarray Platform
Custom miR microarrays were prepared as de- scribed previously.25 Briefly, ~900 DNA oligonucleotide probes representing miRs were spotted in triplicate on coated microarray slides (Nexterion Slide E; Schott, Mainz, Germany), and 3 to 3.5 µg of total RNA from 38 samples were labeled by ligation of an RNA-linker, p-rCrU-Cy/dye (Cy3 or Cy5; Dharmacon, Lafayette, CO) to the 3’ end. Slides were incubated with the labeled RNA for 12 to 16 hours at 42℃ and then washed twice. Arrays were scanned at a resolution of 10 um, and images were analyzed using SpotReader software (Niles Scientific, Portola Valley, CA). Microarray spots were combined and signals normalized as described previously.25
Normalized signals were compared between carci- noma and adenoma samples to identify miRNAs that could be used to differentiate the groups. The significance of differences in miRNA expression were assessed with 2-sided unpaired t test. To control for multiple hypothesis testing, we applied the Benjamini-Hochberg false dis- covery rate. Selected miRNAs were used to classify samples as carcinoma or adenoma. Confidence intervals for classification accuracy were calculated with the Clopper-Pearson method using the binomial distribution.
qRT-PCR Platform
Twenty-three miRs were chosen for classification (18) and normalization (5) on the basis of the microarray results. Measurements were made by qRT-PCR, as re- cently described32; 17 samples served as the training set (8 ACCs, 9 ACAs) and 15 as the validation set (9 ACCs, 4 ACAs, and 2 recurrent carcinomas). Briefly, 1 µg of RNA was incubated in the presence of poly(A) polymerase (NEB), MnC12, and ATP for 1 hour at 37℃. Reverse transcription was then performed on total RNA with an
oligo-dT primer harboring a consensus sequence (Super- Script II RT; Invitrogen, Carlsbad, CA), and the cDNA was amplified by real-time PCR. The latter reaction contained a miR-specific forward primer, a TaqMan probe complementary to the specific miR sequence as well as to part of the polyA adapter sequence, and a universal reverse primer complementary to the consensus sequence of the oligo-dT tail. The cycle threshold (Ct), representing expression levels in logarithmic scale, was determined for each well. Expression was normalized using the mean Ct of the 5 normalizing miRs.
Correlation Between miR Expression and Tumor Characteristics/Weiss Criteria
Spearman correlation and its corresponding P value were calculated for each miR-parameter pair, using the 23 miRs chosen for the PCR experiment and 13 tumor or clinical parameters (weight, diameter, the 9 Weiss criteria, histopathologic diagnosis according to the Weiss system, and patient outcome). The correlation was based on the array data. Fisher combined probability test was used to calculate the P value for the binary parameters, and Yates squared test was used for the nonbinary parameters.
RESULTS
Clinical Findings
According to the Weiss system, 17 of the 46 primary ACTs were diagnosed as ACCs and 29 were diagnosed as ACAs. A summary of the patient characteristics and the clinical and histopathologic details of the primary tumors is
presented in Table 1. Patients with ACAs were followed for 5 to 15 years, and patients with ACCs for 2 to 25 years.
Differentially Expressed miRs in Adrenal Tumors
Of the 38 tumor samples hybridized on microarray, 33 yielded successful readings (8 ACCs, 25 ACAs). Mi- croarray analysis revealed large and highly significant differences between ACCs and ACAs. Over a dozen miRs were upregulated or downregulated in ACCs compared with ACAs, for a false discovery rate of 0.1, with 4- to 50- fold differences (Fig. 1). Upregulation of miR-503 in ACCs was the best single discriminator from ACAs, which had very low or no miR-503 expression (accuracy 97%; 95% confidence interval, 0.842-0.999) (Fig. 2A). Only 1 ACC (case DD-7179) showed low miR-503 ex- pression. A combination of 2 miRs, miR-34a, and miR- 497, both underexpressed in ACCs compared with ACAs, discriminated between the 2 tumor types with 100% sensitivity and 96% specificity (accuracy 97%; 95% confidence interval, 0.842-0.999) (Fig. 3A). One adenoma (case DD-7225) showed low expression of both miRs, similar to the carcinomas, and 1 carcinoma (case DD- 7179) was an outlier of this distribution.
The qRT-PCR platform was used in 17 of the tested samples (8 ACCs, 9 ACAs; training set) to measure the expression of 24 miRs of interest selected from the chip analysis and to set thresholds for the distinction between ACCs and ACAs. Similar separation patterns to those found by microarray study were demonstrated for miR- 503 and for the combination of miR-34a and miR-497 (Figs. 2, 3). In addition, case DD-7179 showed borderline
| TABLE 1. Patient Characteristics and Histopathologic Features of Primary ACAs and ACCs | |||
|---|---|---|---|
| Characteristics | Adrenocortical Adenomas (n = 29) | Adrenocortical Carcinomas (n = 17) | P* |
| Sex (male/female) | 12/17 | 3/14 | NS |
| Age: 0-9, 10-19, 20-39, 40-59, 60-79 | 0, 1, 1, 14,13 | 3, 0, 5, 7, 2 | 0.056 (Y) |
| Side (right/left) | 10/18+ | 5/8+ | NS |
| Weight > 31.5g/≤31.5g | 6/14+ | 10/2+ | 0.009 (F) |
| Diameter > 4/≤4cm | 7/22 | 14/1+ | 1.4e-005 (F) |
| Nuclear grade (0-3) | 3, 5, 20, 1 | 0, 0, 2, 15 | 3.5e-006 (Y) |
| Mitotic rate (low/high) | 29/0 | 4/13 | 2.3e-008 (F) |
| Atypical mitoses (absent/present) | 29/0 | 7/10 | 4.8e-006 (F) |
| Eosinophilic cytoplasm (< 75%/ ≥ 75%) | 26/3 | 0/17 | 6.5e-010 (F) |
| Architecture (not diffuse/diffuse growth) | 28/1 | 3/14 | 3.9e-008 (F) |
| Necrosis (absent/present) | 28/0± | 3/14 | 4.1-009 (F) |
| Venous invasion (absent/present) | 29/0 | 13/4 | 0.015 (F) |
| Sinus invasion (absent/present) | 26/3 | 8/9 | 0.004 (F) |
| Capsular invasion (absent/present) | 27/2 | 4/13 | 1.9-006 (F) |
| Lymph node metastasis (absent/present) | 0 | 0 | |
| Distant metastasis (absent/present) | 26/0+ | 9/7+ | 0.0004 (F) |
| Local recurrence (absent/present) | 26/1+ | 12/2+ | NS (F) |
| Outcomes | |||
| Alive-NED | 28 | 11 | NS (Y) |
| Dead-NED | 1 | 1 | |
| Alive-WD | 2 | ||
| DOD | 3 | ||
* P values are calculated using Fisher (F) or Yates (Y) test for binary and nonbinary parameters, respectively.
+Missing data.
¿Not evaluated in one tumor which underwent preoperative embolization.
ACA indicates adrenocortical adenomas; ACC, adrenocortical carcinomas; DOD, dead of disease; NED, no evidence of disease; WD, with disease.
Microarray: Carcinoma vs Adenoma
+ Not tested
· Significant p<0.042
· Differential (f-change>2)
10
Selected for PCR
Normalizer in PCR Factorlines (x 1.5)
hsa-miR-22
hsa-let-7d
hsa-miR-10b
Carcinoma (n=8)
hsa-miR-125b
10
hsa-miR-99a
hsa-miR-151-5p
hsa-miR-34a
hsa-miR-503
·hsa-miR-30c
0
hsa-miR-483-3p
hsa-miR-195
hsa-miR-210 IU
&
hsa-miR-483-5p
hsa-miR-222
hsa-miR-513a-5p
hsa-miR-497
0
hsa-miR-487a
hsa-miR-214
hsa-miR-29c*
hsa-miR-15a
10
hsa-miR-335
hsa-miR-345
10
hsa-miR-708
10
104
105
Adenoma (n=25)
expression of all 3 miRs (Figs. 2B, 3B), and 1 ACC (case DD-7190) exhibited an adenoma-like signal of miR-34a and miR-497. The selected thresholds were validated on a test set of 15 independent samples (9 ACCs, 4 ACAs, and 2 recurrent carcinomas). Results showed that miR-503 had 100% sensitivity and 100% specificity for dis- tinguishing ACC from ACA, and the combination of miR-34a and mir-497 had an 89% sensitivity and 100% specificity with the same thresholds (Figs. 2C, 3C).
Correlation of miR Expression in ACTs With Clinical Parameters and Weiss Criteria Tumor Weight and Diameter
Statistically significant correlations were found be- tween tumor weight and diameter and expression of miR- 214, miR-125b, and miR-335 (Table 2). Heavier tumors were associated with downregulation of miR-214, miR- 125b, and miR-335. Larger tumors were associated with upregulation of miR-483-3p, miR-483-5p, miR-513a-5p, and miR-503 and downregulation of miR-497, miR-22, miR-214, miR-125b, miR-29c, miR-195, and miR-335.
Weiss Criteria and Diagnosis
A positive and statistically significant correlation to the Weiss criteria was found for miR-503, associated with 9 criteria, miR-483-3p and miR-483-5p, both associated with 8 criteria, and miR-210, associated with 7 criteria (Table 2). Several miRs were correlated with a smaller
number of Weiss criteria. All these miRs were sig- nificantly upregulated in tumors that met multiple Weiss criteria, indicative of malignancy. The following miRs had a negative statistically significant relationship to most of the Weiss criteria: miR-497, miR-214, miR-708, miR-29c, miR-195, and miR-335 (negatively related to 7 criteria each), and miR-99a and miR-125b (negatively related to 6 criteria each). Other miRs were associated with fewer criteria. All of these miRs were significantly downregulated in tumors that met multiple Weiss criteria, indicative of malignancy. Similar significant correlations were noted between miR overexpression or under- expression and tumor diagnosis based on the Weiss criteria. Full details are presented in Table 2.
Outstanding Cases
In general, there was a high level of correspondence between miR expression and the Weiss system. However, 3 tumors had unusual features. Two tumors (cases DD- 7179 and DD-7190) that were originally diagnosed as ACAs and rediagnosed as ACCs using the Weiss system were found to have a miR profile more characteristic of ACA (DD-7179) or very borderline ACC (DD-7190). Both affected patients had an excellent outcome, with no evidence of recurrence or metastasis in 10 and 6 years of follow-up, respectively. Case DD-7179 met 3 of the Weiss criteria and also had macroscopic features of ACC (weight 250g, diameter 9cm). Histopathologically, it was characterized by prominent nuclear pleomorphism,
A
Microarray
ACA
hsa-miR-503
10
ACC
10°
DD-7190
102
DD-7225
DD-7179
0
5
10
15
20
25
30
B
PCR Training set
22
hsa-miR-503
20
18
DD-7190
16
DD-7179
ACA
14
ACC
0
2
4
6
8
10
12
14
16
18
C
22
PCR Validation set
hsa-miR-503
20
18
16
ACA
ACC
14
Recurrence
0
2
4
6
8
10
12
14
16
Ranking
diffuse growth pattern, and eosinophilic cytoplasm; all features of oncocytic ACT (Fig. 4). There was a low ad- enoma-like expression pattern of miR-503 and a border- line carcinoma-like expression of miR-497 and miR-34a. Case DD-7190 also met 3 of the Weiss criteria but had macroscopic features compatible with ACA (weight 40 g, diameter 3 cm). Histopathologically, the tumor had an overall eosinophilic cytoplasm; very few sections showed focal necrosis and increased mitotic activity (Fig. 5). The miR profile was compatible with ACC. A third tumor (case DD-7225) was originally diagnosed to be of un- certain malignant potential and rediagnosed as an ad- enoma using Weiss protocol. It was found to have a miR profile partially characteristic of ACC, namely, down- regulation of both miR-497 and miR-34a. However, miR- 503 expression was low, which is not true for ACC. On
A
Microarray
40000
ACA
hsa-miR-34a
20000
ACC
10000
0
DD-7190
DD-7179
0
0
4000
DD-7225
10
103
10
B
PCR Training set
24
ACA
hsa-miR-34a
ACC
22
8
DD-7190DD-7179
20
18
15
16
17
18
19
20
21
22
23
C
PCR Validation set
24
ACA
hsa-miR-34a
ACC
22
Recurrence
20
18
15
16
17
18
19
20
21
22
23
hsa-miR-497
analysis, we found that the tumor was an ACA according to the Weiss system because it had undergone emboliza- tion culminating in necrosis, which had not been con- sidered in the evaluation. Thus, it actually met only 2 of the 9 Weiss criteria: diffuse architecture and predom- inantly eosinophilic cytoplasm (Fig. 6). The tumor re- curred locally after surgery (compatible with ACC) and was treated both surgically and hormonally. At present, 9 years after the initial diagnosis, the patient is alive and well.
Correlation of miR Expression With Clinical Outcome
Multiple miRs were related to clinical outcome. Positive statistically significant correlations were found for miR-483-3p, miR-483-5p, miR-503, miR-10b, and miR-513a-5p, all of which were overexpressed in patients
| TABLE 2. Statistical Correlations of MicroRNAs With Biological Behavior and Tumor Macroscopic and Microscopic Features | |||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MicroRNAs | Wt | D | Nucl Grade | Mitosis Rate | Mitosis A | Cytoplasm | Arch | Necrosis | Venous Invasion | Sinus Invasion | Capsular Invasion | His/B/M | Local rec | Dist metast | Outcomes |
| 222 r | |||||||||||||||
| P | |||||||||||||||
| Let-7d r | |||||||||||||||
| P | |||||||||||||||
| 487a r | 0.455 | 0.471 | 0.395 | 0.428 | |||||||||||
| P | 0.011 | 0.007 | 0.025 | 0.021 | |||||||||||
| 483-3p r | 0.579 | 0.516 | 0.486 | 0.533 | 0.659 | 0.679 | 0.808 | 0.493 | 0.671 | 0.671 | 0.637 | ||||
| P | 0.0005 | 0.002 | 0.005 | 0.002 | 0.00004 | 0.00002 | 0 | 0.004 | 0.000026 | 2.6e-05 | 0.00009 | ||||
| 497 r | -0.44 | 0.608 | -0.531 | -0.476 | -0.688 | -0.61 | -0.725 | -0.379 | -0.637 | -0.55 | -0.399 | ||||
| P | 0.012 | 0.0002 | 0.002 | 0.006 | 0.00001 | 0.0002 | 0.000004 | 0.032 | 0.00009 | 0.002 | 0.023 | ||||
| 34a r | -0.528 | -0.544 | -0.44 | -0.392 | -0.469 | -0.502 | -0.46 | ||||||||
| P | 0.002 | 0.001 | 0.013 | 0.026 | 0.007 | 0.0055 | 0.008 | ||||||||
| 22 r | -0.394 | ||||||||||||||
| P | 0.026 | ||||||||||||||
| 151-5p r | |||||||||||||||
| P | |||||||||||||||
| 214 r | -0.474 | -0.435 | -0.471 | -0.409 | -0.502 | -0.405 | -0.612 | -0.378 | -0.438 | -0.61 | -0.574 | -0.448 | |||
| P | 0.022 | 0.012 | 0.0065 | 0.02 | 0.003 | 0.021 | 0.0002 | 0.033 | 0.012 | 0.00021 | 0.001 | 0.01 | |||
| 99a r | -0.443 | -0.461 | -0.517 | -0.569 | -0.595 | -0.539 | -0.532 | -0.478 | -0.421 | ||||||
| P | 0.011 | 0.008 | 0.002 | 0.00068 | 0.0004 | 0.001 | 0.0017 | 0.009 | 0.016 | ||||||
| 125b r | -0.472 | -0.376 | -0.499 | -0.358 | -0.524 | -0.52 | -0.587 | -0.43 | -0.571 | -0.478 | -0.349 | ||||
| P | 0.023 | 0.034 | 0.004 | 0.044 | 0.002 | 0.002 | 0.00052 | 0.014 | 0.00065 | 0.009 | 0.05 | ||||
| 708 r | -0.577 | -0.458 | -0.403 | -0.491 | -0.564 | -0.591 | -0.356 | -0.615 | -0.442 | ||||||
| P | 0.0005 | 0.008 | 0.022 | 0.004 | 0.0008 | 0.00046 | 0.0455 | 0.00018 | 0.016 | ||||||
| 483-5p r | 0.571 | 0.404 | 0.406 | 0.481 | 0.596 | 0.651 | 0.738 | 0.444 | 0.562 | 0.562 | 0.42 | 0.776 | 0.553 | ||
| P | 0.00064 | 0.022 | 0.021 | 0.005 | 0.0003 | 0.00005 | 0.000002 | 0.011 | 0.0008 | 0.00082 | 0.02 | 0.000001 | 0.001 | ||
| 29c r | -0.427 | -0.459 | -0.475 | -0.466 | -0.631 | -0.528 | -0.673 | -0.449 | -0.52 | -0.454 | |||||
| P | 0.015 | 0.008 | 0.006 | 0.007 | 0.0001 | 0.002 | 0.00003 | 0.01 | 0.002 | 0.013 | |||||
| 195 r | -0.389 | -0.473 | -0.527 | -0.481 | -0.616 | -0.528 | -0.707 | -0.399 | -0.586 | -0.55 | -0.41 | ||||
| P | 0.028 | 0.006 | 0.002 | 0.005 | 0.00012 | 0.002 | 0.000009 | 0.024 | 0.00042 | 0.002 | 0.019 | ||||
| 30c r | -0.358 | -0.364 | -0.349 | ||||||||||||
| P | 0.044 | 0.041 | 0.05 | ||||||||||||
| 503 r | 0.395 | 0.459 | 0.699 | 0.592 | 0.652 | 0.604 | 0.873 | 0.413 | 0.348 | 0.609 | 0.757 | 0.685 | 0.513 | ||
| P | 0.025 | 0.008 | 0.000009 | 0.0003 | 0.00005 | 0.0002 | 0 | 0.019 | 0.05 | 0.002 | 0.000001 | 0.00004 | 0.002 | ||
| 10b r | 0.414 | 0.399 | 0.526 | 0.374 | |||||||||||
| P | 0.021 | 0.024 | 0.003 | 0.035 | |||||||||||
| 15a r | -0.388 | -0.364 | -0.36 | -0.586 | -0.469 | ||||||||||
| P | 0.031 | 0.041 | 0.043 | 0.0008 | 0.007 | ||||||||||
| 210 r | 0.413 | 0.428 | 0.438 | 0.502 | 0.479 | 0.511 | 0.367 | 0.367 | 0.434 | ||||||
| P | 0.019 | 0.014 | 0.012 | 0.003 | 0.0055 | 0.003 | 0.0385 | 0.039 | 0.019 | ||||||
| 335 r | -0.437 | -0.542 | 0.551 | -0.528 | -0.461 | -0.703 | -0.677 | -0.678 | -0.532 | -0.705 | -0.527 | -0.39 | |||
| P | 0.037 | 0.001 | 0.001 | 0.012 | 0.008 | 0.000007 | 0.000021 | 0.000028 | 0.001 | 0.000007 | 0.003 | 0.027 | |||
| 345 r | -0.521 | -0.435 | |||||||||||||
| P | 0.002 | 0.013 | |||||||||||||
| 513a-5p r | 0.414 | 0.391 | 0.366 | 0.402 | 0.45 | ||||||||||
| P | 0.0185 | 0.027 | 0.04 | 0.022 | 0.01 | ||||||||||
A indicates atypical; Arch, architecture; B, benign; D, diameter; Dist metast, distant metastases; His/B/M, histologic diagnosis according to Weiss system; M, malignant; Nucl, nuclear; rec, recurrence; Wt, weight.
with a detrimental outcome (alive with disease or dead of disease). Negative statistically significant correlations with clinical outcome were found for miR-497, miR-34a, miR-214, miR-99a, miR-125b, miR-195, miR-30c, miR- 15a, miR-335, and miR-345, all of which were underex- pressed in patients with a detrimental outcome. Similarly, miR-487a, miR-483-5p, miR-503, miR-10b, and miR-210 were significantly overexpressed in tumors associated with distant metastases, and miR-497, miR-34a, miR-214, miR-99a, miR-125b, miR-708, miR-29c, miR-195, miR- 15a, and miR-335 were significantly underexpressed in tumors associated with distant metastases (Table 2).
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DISCUSSION
The pattern of miR expression has been found to be specific for tumor type, making it an important biological factor in the identification of cancers of unknown origin and in the differentiation of malignancies arising in the same organ.23-25,27,28,33 The present study examined the possibility that miR expression patterns may also help pathologists and clinicians distinguish benign from ma- lignant tumors of the same organ; specifically, ACCs and ACAs, which are often similar morphologically.
The results clearly demonstrated that a large number of miRs are differentially upregulated or downregulated in ACTs and can serve to corroborate or negate diagnoses made according to the Weiss system. Similar to other studies, we found that miR-335, miR-195, and miR-497 were significantly downregulated in ACCs compared with ACAs.10,29,30 Interestingly, a common chromosomal ACC deletion, 17p13,34,35 contains the genomic cluster of miR- 195 and miR-497.10 Moreover, miR-335 is downregulated also in breast cancer,36 where it was found to be a tumor- suppressor gene,37 and miR-195 is downregulated in squ- amous cell carcinoma of the tongue38 and chronic lymphatic leukemia.39
In the present study, miR-503 was the best single discriminator between ACTs, with prominent over- expression in ACCs compared with ACAs. Similar over- expression in ACCs was reported by Tömböl et al31 and Soon et al,30 and it has also been described in retino- blastoma40 and parathyroid carcinoma.41 These findings are compatible with earlier reports in which miR-503 di- rectly targets cell cycle arrest and differentiation genes.10 Patterson et al,29 in a study of ACCs, reported upregu- lation of miR-483-5p, which is related to the expression
of insulin growth factor 2, a hormone typically overexpressed in ACC.17,29 Accordingly, we found that the second arm of the same pre-miR, miR-483-3p, was upre- gulated in ACC, as reported also by Doghman et al.28 In addition, miR-483-3p is upregulated in Wilms tumor and carcinomas of the breast, large bowel, and liver.42 Others showed that inhibition of miR-483-3p or miR-483-5p in the human ACC cell line NCI-H295R caused a significant re- duction in cell proliferation: Cells that were transfected with anti-miR-483-5p had a significant increase in apoptosis.28 Another miR that was overexpressed in ACCs in our study was mir-210. This finding was also noted by Özata et al,10 who suggested that because the expression of mir-210 is regulated by hypoxia-inducible factor la (HIF1x), it may be involved in tumor hypoxia, an important factor in radio- therapy and chemotherapy resistance. Moreover, mR-210 was found to play a role in a wide range of cell processes, including proliferation, apoptosis, and differentiation, in addition to DNA repair and cell metabolism.43 Its ex- pression is known to be elevated in human solid tumors, and its overexpression in tissue has been significantly associated with poor prognosis of various types of carcinoma.44
We observed that the combination of 2 miRs, miR- 497 and miR-34a, which are downregulated in ACCs rel- ative to ACAs, discriminated well between the 2 types of tumors. Both these miRs are also often downregulated in carcinomas and have been shown to promote cell apopto- sis.45,46 Specifically, miR-497 is underexpressed in child- hood ACT,28 peritoneal carcinoma,45 and male breast cancer.47 Overexpression of miR-497 and miR-195 in the human ACC cell line NCI-H295R was associated with a significant decrease in cell growth and induction of cell death by apoptosis.10
It is noteworthy that the method of diagnosing ACTs as benign or malignant differed in the above- mentioned studies.10,29-31 Soon et al,30 as in the present study, applied the Weiss protocol. Patterson et al29 di- agnosed ACCs by the presence of gross local invasion and/or metastasis at presentation or during follow-up, and ACAs, by lack of evidence of recurrence at follow-up (mean 2.1y). Özata et al10 used the World Health Organization classification, and Tömböl et al31 did not report the method of diagnosis.
The high level of correspondence in the present study between miR expression and the Weiss system is an important finding that lends validity to the Weiss system. However, there were 3 exceptions: 2 tumors originally diagnosed as ACAs and reclassified as ACCs with the Weiss system had a miR profile more compatible with ACA or very borderline ACC. Both patients had an ex- cellent outcome. Similarly, in the study of Soon et al,30 1 tumor diagnosed as ACC according to the Weiss system was found to have the miR profile of ACA. The authors suggested that the tumor could be considered a false- positive by the Weiss system, and that miR analysis may be as sensitive as and more specific than the Weiss system for predicting the biological behavior of ACTs. A third tumor in our study that was initially diagnosed to be of uncertain malignant potential and then reclassified as an
ACA was found to have a miR profile more characteristic of ACC. The tumor recurred following surgery, but the patient is well 9 years from the initial diagnosis. These 3 tumors are unusual in that they appear to be in- determinate in terms of both their histopathologic fea- tures and their miR expression pattern. This raises the possibility of the existence of true borderline ACTs.
Given that the aberrant overexpression or under- expression of many of the miRs was related to the criteria that define malignancy as part of the Weiss system and that the miRs evaluated have been associated with a variety of malignant processes in the literature, we speculate that the miRs identified here are not specific to ACTs but rather represent a functional “signature” of malignancy in general. At the same time, the miRs associated with Weiss criteria showed a similar statistically significant association with tumor weight and diameter, the traditional macroscopic parameters used in the diagnosis of ACTs, supporting this basic, time-honored method of evaluating ACTs.
The study investigators who revised the tumors before patient recruitment to the study were blinded to all the clinical details, including patient age. Thus, although some studies suggest that ACTs should be approached differently in children and adults due to the more favor- able prognosis in children, at least in the infantile group, 48 in our study, tumors in all age groups were evaluated in the same manner. Three tumors in children, aged 1, 2, and 8 years at diagnosis, were compatible with ACC accord- ing to both Weiss criteria and their miR profile. All 3 patients are alive with no evidence of disease, including the child who had liver and lung metastases.
The miR expression profile of the ACTs was sig- nificantly associated with survival and as such, was found to be a strong prognostic indicator. Analysis of the disease- specific survival yielded statistically significant correlations with about 18 miRs. Poor outcome was associated with downregulation of miR-195 and upregulation of miR-483- 5p, similar to the finding of Soon et al,30 and with over- expression of miR-503. In addition, Özata et al10 identified overexpression of miR-1202 and miR-1275 as an indicator of detrimental outcome. Other miRs in the present study that were associated with a detrimental outcome when overexpressed were miR-483-3p, miR-483-5p, miR-10b, and miR-513-5p, and when underexpressed, miR-497, miR- 34a, miR-214, miR-99a, miR-125b, miR-195, and miR-30c. Not surprisingly, the same miRs that differentiated ACC from ACA were also related to patient outcome.
In conclusion, the results of our study, as well as others,10,29-30 suggest that miR profile evaluation may serve as another tool in the diagnostic arsenal of ACT. The strong correlation between the miR expression profile and the histopathologic Weiss system supports the val- idity of the Weiss system and suggests that both these disparate methods accurately identify malignant ACTs. However, in borderline ACTs and ACTs with artifactual changes or other factors that impede complete analysis, the addition of miR evaluation may greatly assist clini- cians in predicting the biological behavior of the tumor and in selecting the appropriate treatment accordingly.
ACKNOWLEDGMENTS
The authors thank Gloria Ginzach and Melanie Kawe for their editorial and secretarial assistance.
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