Molecular Profiling of Matched Samples Identifies Biomarkers of Papillary Thyroid Carcinoma Lymph Node Metastasis

Research Article

Janete M. Cerutti,1,2 Gisele Oler,2 Pedro Michaluart, Jr.,4 Rosana Delcelo,3 Robert M. Beaty,1 Jennifer Shoemaker,5 and Gregory J. Riggins1

1Department of Neurosurgery, Johns Hopkins University Medical School, Baltimore, Maryland; 2Genetic Bases of Thyroid Tumors Laboratory, Division of Genetics, 3Department of Pathology, Federal University of Saõ Paulo, and 4Division of Head and Neck Surgery, Department of Surgery, University of SaõPaulo Medical School, Saõ Paulo, SP, Brazil; and 5Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, North Carolina

Abstract aggressive PTC phenotype would help improve individualized Biomarkers of papillary thyroid carcinoma (PTC) metastasis treatment for this cancer. Several groups have done expression profile of metastatic can accurately identify metastatic cells and aggressive tumor in vitro behavior. To find new markers, serial analysis of gene primary PTC or model systems to identify clinical outcome expression (SAGE) was done on three samples from the same markers (5, 6). Although these approaches could identify useful patient: normal thyroid tissue, primary PTC, and a PTC lymph prognostic markers, they do not directly identify gene expression node metastasis. This genomewide expression analysis iden- changes that occur in the metastatic cells. Identification of genes tified 31 genes expressed in lymph node metastasis, but not that are consistently expressed in metastatic PTC cells could yield in the primary tumor. Eleven genes were evaluated by useful biomarkers. These markers have potential prognostic use quantitative real-time reverse transcription-PCR (qPCR) on and may also help identify occult metastatic cells in lymph node independent sets of matched samples to find genes that were biopsies. Eventually, they could also be investigated as serum consistently different between the tumor and metastatic markers. In addition to the practical uses as markers, genes samples. LIMD2 and PTPRC (CD45) showed a statistically associated with metastasis could help reveal the molecular significant difference in expression between tumor and mechanisms of the metastatic process. metastatic samples (P < 0.0045), and an additional gene To identify gene expression changes that occur subsequent (LTB) had borderline significance. PTPRC and LTB were tested to thyroid LNM, we did SAGE on matched normal thyroid (NT), by immunohistochemistry in an independent set of paired primary PTC, and its LNM and in a normal lymph node. Serial samples, with both markers showing a difference in protein analysis of gene expression (SAGE; ref. 7) was employed because expression. All 20 metastases from 6 patients showed of its ability to accurately produce comprehensive expression pro- expression in both markers, with little or no expression in files from small samples and because there are archived databases primary tumor. Some of these markers could provide an of SAGE expression profiles of human tissues freely available for improved means to detect metastatic PTC cells during initial comparison. staging of a newly diagnosed carcinoma and/or to rule out To our knowledge, this is the first gene expression comparison recurrence. The functional role of these genes may also on matched NT, primary PTC, and metastasis samples. We provide insight into mechanisms of thyroid cancer metastasis. identified transcripts exclusively expressed in a LNM library [Cancer Res 2007;67(16):7885–92] and, therefore, potentially related to the metastatic process of PTC. The expression of the selected transcripts were investigated in a series of matched-normal, primary tumor and LNMs by Introduction real-time reverse transcription-PCR (qPCR). The transcripts that Papillary thyroid carcinoma (PTC) is the most common thy- were found consistently overexpressed in LNMs were evaluated roid cancer, accounting for about 80% of all thyroid cancers. by immunohistochemistry in an independent set of paired sam- Although PTCs are usually curable with standard surgical and ples for confirmation. The markers located in this study may have adjuvant radioiodine treatment, neck lymph node metastases eventual utility for better prediction and detection of PTC (LNM) are found in 30% to 65% of cases at initial diagnosis metastasis. (1–3). Unfortunately, about 15% of cases with LNMs also display a very aggressive behavior, characterized by local invasion, distant metastasis, treatment resistance, and increased mortality. Materials and Methods Due to this clinical heterogeneity, the management of PTC is Generation of SAGE libraries. Matched tissues of a NT, a PTC, and often controversial and depends on the detection of distant its LNM were chosen for SAGE (Table 1, case 1). This matched set metastasis (2–4). An earlier and more accurate detection of the was chosen in part because the sample quality was high, measured by the high percentage of tumor cells observed by H&E histopathology done on frozen sections from primary and metastatic tumors. The primary sample was from the tumor core, in an attempt to avoid the capsule and surrounding normal tissue. To eliminate the expression of normal lymph node cells without any metastasis, a SAGE library was generated from Requests for reprints: Janete Cerutti, Rua Pedro de Toledo 781, 12j andar., Federal a normal lymph node (NL; Stratagene). The libraries were constructed University of Sa˜oPaulo, 04039-032, Sa˜oPaulo, SP, Brazil. Phone: 55-11-5081-5233; using NlaIII as the anchoring enzyme as described in the original SAGE Fax: 55-11-5084-5231; E-mail: [email protected]. I2007 American Association for Cancer Research. procedure (7), and the ditag containing plasmid inserts were sequenced doi:10.1158/0008-5472.CAN-06-4771 through the SAGE portion of the Cancer Genome Anatomy Project (8). Tags www.aacrjournals.org 7885 Cancer Res 2007; 67: (16).August 15, 2007

Table 1. Summary of clinical data

Cases Diagnosis Sex Age at Nodule Extrathyroidal Metastasis at Metastasis Persistent Iodine BRAF mutation diagnosis (y) size (cm) extension presentation (follow-up) disease LNMs

First validation set c 1 PTC* , F 19 3.5 N Y Y Y Y V600E + K601del c 2FVPTC F 76 1.0 N Y Y Y Y V600E c 3 PTC M 41 1.5 Y Y NA Y Y V600E c 4 PTC F 55 2.5 Y Y N N Y V600E c 5 FVPTC F525.0Y Y N NY N c 6 PTC F 29 3.5 Y Y NA Y Y V600E c 7 PTC F 39 NA Y Y NA NA NA V600E + K601del c 8 FVPTC F 49 2.0 Y Y Y (lung) Y Y N c 9 PTC F 49 2.2 Y Y NA Y Y ND b 10 PTC F 46 1.7 Y Y N N Y V600E b 11 PTC F 421.5 Y Y NA Y Y V600E 12FVPTC x F 15 4.5 Y Y NA NA NA N Second validation set c 13 PTC M 34 5.5 Y Y Y N Y V600E + K601del c 14 PTC M 622 .8 Y Y Y YY ND c k 15 PTC , M 34 0.7 Y Y Y YY ND c k 16 FVPTC , M 23 1.3 Y Y Y YY ND c k 17 PTC , F 19 3.5 N Y Y YY ND 18 PTCx F 33 1.2N Y N NY ND

Abbreviations: FVPTC, follicular variant of PTC; NA, not available. Y, yes; N, no. *Case 1 was chosen for SAGE. cMatched normal, tumor, and lymph node metastasis. bMatched normal and lymph node metastasis. x Matched tumor and lymph node metastasis. kLymph node metastases obtained during reoperation for recurrence.

were extracted from sequence text files and processed to remove duplicate 2paired NT and LNM (Table 1, first validation set). Additionally, a normal ditags, linker sequences, and repetitive tags using SAGE 2002 software lymph node was included as a negative control. version 4.12.6,7 For further confirmation of metastasis genes, a second validation set of SAGE analysis. The metastasis-derived library was compared with paraffin-embedded sections was obtained from six matched samples of primary tumor using SAGE 2000 software. Pairwise and Monte Carlo normal lymph nodes, normal thyroid, primary tumor, and LNM (Table 1, simulations were used to identify transcripts in which difference was second validation set). The follow-up of these patients showed that three out statistically different at a P value of V0.001. Transcripts that were of six patients had regional recurrence (Table 1, cases 15–17). There were at overexpressed in the metastasis library when compared with primary least two lymph nodes with metastasis studied from each case in the second tumor and, therefore, candidate genes as associated with metastasis validation set. Additionally, in the three patients who recurred at least two process, were assessed in LNM and LN libraries. more lymph nodes were studied from the recurrence. A total of 20 LNMs Tissue samples for validation of candidate genes. To test our were investigated. A third set of samples included 15 primary classic and hypothesis, the metastasis candidate genes were first analyzed in a series of follicular variant of PTC (metastatic and nonmetastatic tumors, not shown matched normal thyroid tissue, primary PTC, and LNM provided by the in Table 2). The paraffin-embedded sections were selected from the archives Hospital das Clıńicas, Universidade de Saõ Paulo. The neck dissection was of the Department of Pathology, Federal University of Saõ Paulo. The study done by a single surgeon (P.M.) during thyroidectomy procedure. All was approved by the Ethic and Research Committees from both universities samples were collected and frozen immediately after surgical biopsy and and was conducted in accordance with the Declaration of Helsinki stored at 80jC. Principles. Tissue histology of H&E-stained, paraffin-embedded sections obtained RNA isolation, cDNA synthesis, and qPCR. Total RNA was isolated by from frozen samples was evaluated by an experienced pathologist (R.D.) TRIzol (Invitrogen Corp.). About 1 Ag of total RNA was treated with a DNase and used to confirm the initial diagnosis (Table 1). This histology was also (Ambion) and was reverse transcribed to cDNA using Super-Script II used to determine the percentage of tumor cells, and only cases containing Reverse Transcriptase kit with an oligo(dT)12–18 primer and 10 units of a high percentage of tumor cells in both primary tumor and metastasis were RNase inhibitor (Invitrogen Corp.). An aliquot of cDNA was used in 20 AL considered for validation. Using this criterion, 12series were selected: PCR reactions containing TaqMan Universal PCR Master mix, 10 Amol/L of 9 matched normal thyroid, PTC, and LNM; 1 paired PTC and LNM; and each specific primer and FAM-labeled probes for the target genes or reference gene (QP-C), and VIC-labeled probe for the second reference gene (RS8; TaqMan Gene Assays on Demand; Applied Biosystems). Quantitative PCR reactions were done in triplicate, the threshold cycle

6 (C ) was obtained using the Applied Biosystem software and were averaged Available at http://www.sagenet.org. t 7 V The full set of tag counts for all four libraries is available for downloading or (SD 1). Relative expression levels were calculated according to the formula (Ce Re) (Cn Rn) C analysis at the SAGE Genie Web site at http://cgap.nci.nih.gov/SAGE (9). 2 /2 , where Ce is the t cycle number observed in the

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experimental sample for controls genes, Re is the Ct cycle number observed samples. The second objective was to determine whether a class predictor C in the experimental sample for the reference gene, Cn is the average t cycle (tumor versus metastasis) could be developed using the qPCR data from number observed in the normal thyroid tissues for control genes, and Rn is these 11 genes. To investigate the development of an expression- the average Ct cycle number observed in the normal thyroid tissues for the based predictor that could be used to predict tumor or metastatic class, reference gene (10, 11). The results obtained from relative expression levels we followed the framework outlined by Radmacher et al. (12) using were log transformed and used for statistical analysis. Representative results the compound covariate predictor for gene expression data (12, 13). are shown in Fig. 1. The performance of the predictor was tested using leave-one-out cross- Statistical analysis. The first objective of this analysis was to determine if validation for all steps of the prediction procedure (i.e., selection of the expression values, as measured by qPCR, for 11 genes were different differentially express genes as well as creation of the prediction rule; between tumor and metastases (n = 10 pairs) or between normal and refs. 12, 14). We assessed the significance of the performance of the predictor metastases (n = 11 pairs), using paired data (Table 1). The comparison of the using the permutation-based test outlined by Radmacher et al. (12), in which expression levels was carried out using a paired Student’s t test. To correct the class labels were randomly permuted and the proportion of data sets that for multiple tests, a Bonferroni correction was used. A comparison was had a cross-validated error rate and is small as the error rate observed in the designated as statistically significant if the t statistic was found to be data set was calculated (12). The only difference in the permutation test from significant using an a level that had been adjusted (using a Bonferroni that used by Radmacher et al. (12) was that, because the data were paired in adjustment) to keep the family-wise error rate at 0.05. A one-sided test was the present study, we used a paired permutation. In addition, because of the used because of the expectation that the gene expression in the metastatic small size of the data set, we were able to generate the complete permutation samples would be higher than the gene expression in the tumor or normal distribution.

Table 2. Genes induced in LNMs selected for verification and normal thyroid-specific genes

b Tag Normal Normal Primary LNMs* Transcript Aliases GenBank Location Gene ontology c sequence lymph thyroid* tumor* description accession node* no.

GTCAACAGTA 0 0 0 21 ABCC3, ATP-binding MRP3 NM_003786 17q22 Transporter activity cassette, subfamily C (CFTR/MRP), member 3 GCAGTGGGAA 0 0 0 38 LTB , lymphotoxin h TNFC NM_002341 6p21.3 Tumor necrosis factor receptor- binding activity GTAGCGCCTC 0 0 0 9 CST7, cystatin F CMAP BC015507 20p11.21 Cysteine protease inhibitor activity TTAACTGTGT 0 0 0 13 SYT12, synaptotagmin XII SRG1 BC037406 11q13.2Transporter activity CTTTTTTCCC 0 3 1 23 CD48, B-cell BLAST1, BC0161821q21.GPI anchor membrane protein SLAMF2 binding TTAAATCCCA 24 0 29 PTPRC, protein tyrosine LCA, CD45 NM_002838 1q31 Protein tyrosine phosphatase, phosphatase receptor type C activity AAAGCAAAAA 0 4 1 23 PTPN4, protein tyrosine PTPMEG1 NM_002830 2q14.2 Protein tyrosine phosphatase, phosphatase nonreceptor type 4 activity TTTCAATAGA 6 1 1 23 LIMD2 MGC10986 BC004400 17q23.3 Metal ion binding Genes previously associated with papillary metastatic process GAGGCCATCC 4 10 1 26 LSM7, U6 small nRNA No BC018621 19p13.3 RNA binding associated CAATTAAAAT 0 3 25 94 MET proto-oncogene HGFR, NM_000245.2 7q31 Protein tyrosine- RCCP2 kinase activity CAGGCCCCAC 4 17 18 72 S100A11, S100 calcium Calgizzarin BC001410 1q21 Calcium ion binding protein A11 binding Genes involved in thyroid function GATGAATAAA 0 75 0 0 TPO, thyroid peroxidase No M17755 2p25 Thyroid hormone generation CGGTGAAGCA 0 134 16 57 TG, thyroglobulin No NM_003235 8q24.2 Thyroid hormone generation ATGCTAAGAG 0 30 20 DIO2, deiodinase, No NM_000793 14q24.2 Thyroid hormone iodothyronine, type II generation

NOTE: SAGE libraries are posted at http://cgap.nci.nih.gov/SAGE. *SAGE tag counts shown in each column refer to the abundance of SAGE tags in the libraries after normalization to 200,000 total tags. cTranscript description refers to the gene name to which tag was attributed, according to the HUGO/GDB nomenclature committee–approved symbols. bGene classification was by molecular function (http://cgap.nci.nih.gov/Genes/AllAboutGO).

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antibody specificity included incubation with rat immunoglobulin G used at the same concentration as the first antibody. Positive and negative controls were used in each run. All slides were scored in a blinded fashion, with immunopositivity evaluated semiquantitatively as follows: negative () when <10% of the cells were immunoreactive, and positive (+) when more than 10% of cells were immunoreactive. Immunohistochemical staining was evaluated independently by two investigators.

Results Analysis of SAGE data. A total of 481,775 SAGE tags were obtained from four libraries, representing 160,951 unique transcript tags. The number of SAGE tags per library ranged from 99,911 (NL) to 143,689 (PTC). Tag numbers were normalized to 200,000 tags per library. To identify genes potentially involved in metastasis, a comparison between primary tumor and LNM libraries was done. Monte Carlo simulations yielded 498 tags statistically significant at a P value of V0.001 or 319 tags at a P value of V0.0001. A total of 31 of the 498 transcripts were highly expressed in the metastasis library and not expressed or expressed at low levels in primary tumor, whereas 47 transcripts were underexpressed in the metastasis library. To refine our analysis, the transcript expression of the metastasis-associated genes was assessed in the normal lymph node and normal thyroid libraries. Those transcripts with the greatest fold induction in metastasis library were chosen for validation by qPCR because they could not only help better understand the metastatic process, but also had potential as prognostic markers. Additionally, we chose three genes (MET, LSM7, and S100A11) whose expression was previously reported in primary tumors with metastatic potential, although expression within the metastasis was not reported (15–17). Table 2lists the 11 transcripts selected for validation and their tag counts in our four SAGE libraries. For comparison, we show in Table 2the transcript levels for those genes known to be involved in normal thyroid physiology. According to gene ontology databases, the differentially expressed genes are mainly involved in transport and cell signaling. Among the transcripts highly expressed in LNM library and not expressed in primary tumor were six transcripts (CXCR4, SNC73, PRSS1, CXCL13, STAT5A,andSDC1) that were previously associated with invasion and metastases in other cancers. Although not selected for validation by qPCR or immunohistochemistry, Figure 1. Relative levels of expression determined by qPCR in matched SAGE analysis suggested that their expression correlated with samples of normal thyroid (white columns), PTC (gray columns), and LNMs (black columns; samples 1–9), matched normal and metastases (samples metastasis in this patient (P < 0.001). However, further analysis will 10 and 11), matched tumor and metastases (sample 12), and normal lymph be needed to determine a role in LNM of PTC. node (sample 13). Transcript levels were normalized to the average of RS8 and QP-C control genes, which were uniformly expressed in all three thyroid Relative levels of gene expression for selected candidate SAGE libraries. Numbers correspond to cases described in Table 1. The genes. We used qPCR to test the expression of the transcripts qPCR data were log 2 transformed for statistical analysis. The paired t test shown in Table 1 in matched sets of normal thyroid, primary PTC, (normal versus metastases) showed that PTPRC, P = 0.001; LIMD2, P = 0.0016; LTB, P = 0.00096; and CD48, P = 0.00042. ABCC3 (P = 0.0094) and LNMs. This was the first validation set. We compared the was very close to significance. The paired t test (tumor versus metastases) results obtained from SAGE with the qPCR results for the samples showed that PTPRC (P = 0.00059) and LIMD2 (P = 0.00056) expression was used to generate NT, PTC, and LNM libraries (case 1, Table 1 and significantly different at the 0.05 level. Fig. 1). When the initial samples were used, the difference predicted by SAGE was confirmed for all 11 genes. Immunohistochemical analysis. Sections (3 Am) were deparaffinized PTPRC was found overexpressed in all metastases analyzed and rehydrated through a graded series of ethanols. Endogenous peroxidase when compared with paired normal thyroid and primary tumor was quenched using a 3% solution of hydrogen peroxide in methanol for (Fig. 1, samples 1–12) and normal lymph node (Fig. 1, sample 13). 30 min. Steaming retrieval was done in buffer AR-10 (BioGenex) for 10 min LIMD2 and CD48 were consistently expressed in the LNMs and and then allowed to cool for 30 min. The sections were incubated with sample 13 primary antibodies for at least 16 h at 4jC, followed by incubation with were not expressed in the normal lymph node (Fig. 1, ) the labeled polymer DAKO EnVision+ System, HRP (DAKO Laboratories). and not expressed or expressed at very low levels in most of the Hematoxylin was used as the nuclear counterstain. Anti-CD45 (T29/33; matched normal thyroid tissues and/or primary tumors. DAKO Laboratories) was used at a dilution of 1:800, and anti-LTh (FL244; LTB and ABCC3 were remarkably higher in all metastases and Santa Cruz Biotechnologies) was used at a dilution of 1:50. The control for expressed at much lower levels in a number of primary tumors

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2007 American Association for Cancer Research. Predicted Markers for Lymph Node Metastasis tissues (Fig. 1). Although CST7 expression was elevated in the lymph node, which may explain the strong brown staining majority of paired primary tumor, its expression was markedly observed (Fig. 2) and the qPCR results (Fig. 1). To ascertain that higher in the LNMs. Therefore, some genes analyzed here may start PTPRC was found expressed at lower levels in the normal lymph expression earlier than at the onset of metastasis, but still prove nodes, we additionally investigated the expression of PTPRC in six useful as markers of metastasis. normal axillary lymph nodes. Although PTPRC expression was LSM7 and SYT12 showed a similar level of expression in normal found in the lymphocytes in the normal lymph nodes, the number thyroid, primary tumor, and LNM in most samples tested. Contrary of lymphocytes with a positive expression of PTPRC was inferior to results were obtained for PTPN4. The validation data showed that the metastatic lymph node (Fig. 2). We further assessed the PTPN4 was underexpressed in most LNMs rather than overexpressed. expression of PTPRC in 15 primary PTCs (follicular variant and For those genes previously reported to be associated with classic). Immunohistochemistry analysis revealed that 13 out of 15 metastasis, but not implicated in the late stages of metastasis by PTCs were negative for PTPRC. In one case, however, tumor- our SAGE data, qPCR data also showed a lack of overexpression in infiltrating lymphocytes were positive. In the remaining case of the metastases. For example, S100A11 was observed in LNMs, and PTC with a trabecular-insular area, a focal staining was observed in its expression was higher in most primary tumors than in the the epithelial cells. matched metastases. MET was expressed in high levels in about qPCR data showed that LTB was highly expressed in most LNMs, 60% of LNMs analyzed and in intermediate levels in primary although it was expressed at very low levels in a few primary tumors from these cases. In the remaining cases, however, the tumors. Because it was very close to the significance, we tested LTB expression level of MET was higher in primary tumor and normal expression by immunohistochemistry. In the second set of thyroid tissue than in corresponding metastases. Although we did validation, LTB immunoreactivity was positive in all tumors cells not find MET specific for metastasis, our results are in agreement within the lymph node in all 20 metastases analyzed but was not with the literature where MET was found overexpressed in about detected in any adjacent cells (Fig. 2). Additionally, LTB was 60% to 70% of metastatic primary PTC (18, 19). negative in normal lymph nodes and matched normal thyroid Statistical analysis. In the first comparison PTPRC, LIMD2, LTB, tissue and primary tumors (Fig. 2). When LTB was investigated by and CD48 were found to be significantly different between the immunohistochemistry in 15 primary PTC, as suggested by qPCR, normal and metastatic samples (P value <0.0045). PTPRC and 3 out of 15 primary tumors showed a weak staining of LTB in the LIMD2 were found to be statistically significant between tumor and epithelial tumor cells. LTB was negative in the surrounding normal metastatic classes (P value <0.0045). cells. Interestingly, all positive tumors were highly invasive and had The second analysis determined whether a subset of these 11 metastasis to the lymph nodes. genes could be used to predict metastasis versus tumor class. The class predictor used genes whose expression levels were declared significantly different at the 0.05 family-wise error level using the Discussion t test (P value <0.0045). The sample t statistics were used as PTC is the most frequent thyroid carcinoma. Lymph nodes are weights in the compound covariate predictor (12). To evaluate the the most frequent site of PTC metastasis and, if found, predict predictor, we used leave-one-out cross-validation: for each run, one recurrence and poor survival (20). LNMs have also been identified in tumor-metastatic pair of samples was left out, and the predictor the absence of clinically detectable primary tumor. Like most lethal developed on the remaining nine pairs of samples. The two left-out cancers, death from PTC is from metastatic spread rather than samples were predicted. We used all the steps of the prediction invasion of the primary tumor. Therefore, it is of particular procedure, including the selection of differentially expressed genes, importance to quickly identify those patients with aggressive as well as the creation of the prediction rule (14). Using leave-one- disease, so that the patient can be treated before metastatic spread. out cross-validation, 4 of the 20 samples were misclassified for a Over the last decade, several biological markers tested in prediction accuracy of 80% with a 95% two-sided confidence primary tumors have been explored for their value in predicting interval of 0.56, 0.94. To assess the significance of these prediction lymph node involvement in PTC (6, 21–23). Among all candidate results, we implemented a permutation test. The proportion of prognostic markers, BRAF mutation is claimed as one of the most random permutations that classified four or fewer misclassifica- effective markers in predicting clinical outcome in classic PTC. tions was 0.013. Thus, the results of the prediction analysis are However, BRAF mutation as a high-risk marker is controversial. statistically significant. PTPRC and LIMD2 were always selected in First, a number of groups found an association between BRAF each step of the cross-validation procedure (10 out of 10 times, i.e., V600E mutation in PTC and high-risk tumor features (24, 25), and each time a pair of samples was left out). others found no association (26–28). Second, BRAF mutation is Immunohistochemical analysis. To ascertain if our candidate found in about 30% of classic variant of PTC. In the BRAF metastasis-associated markers had increased protein levels in the mutation-negative group of patients, which includes the remaining LNM cells compared with other tissues, immunohistochemistry classic PTC and other variants, one cannot exclude lymph node analysis was done in a second validation set of paired normal lymph involvement or distant metastases. Lastly, we and others have node, normal thyroid, and primary tumor and LNMs (Table 1). Be- identified BRAFde novo mutations in the LNM cells that were cause there is no commercially available antibody for LIMD2, immu- absent in matched primary tumor (29, 30). nohistochemistry analysis was done for PTPRC (CD45) and LTB. Thus far, no clinical markers associated with lymph node or As shown by qPCR, PTPRC, also named CD45 or LCA, was highly distant metastases of thyroid metastasis have reached clinical expressed in LNMs and was not expressed in matched normal practice. A question arising is why the reported prognostic markers thyroid and primary tumors. In the metastatic lymph nodes, a have failed to appear in the clinic. One possible explanation is that strong staining was observed in lymphocytes rather than in the most studies to date have attempted to make prediction from sets metastatic cancer cells. Of note, lymphocytes were the predomi- of primary tumors and have not employed an analysis of matched nant cells in the metastatic lymph node, compared with normal primary/metastatic pairs which can be difficult to obtain clinically. www.aacrjournals.org 7889 Cancer Res 2007; 67: (16).August 15, 2007

Figure 2. Representative results of immunohistochemical analysis. There was no PTPRC reactivity observed in normal thyroid (A) and primary tumors (B and C). Strong brown staining for PTPRC was observed in the surrounding immune cells in metastatic lymph node (D and E). Lymphocytes were positive for PTPRC in normal lymph node (F), but with a different pattern and intensity from that in (E) and (F). No staining for LTB was observed in normal lymph node, normal thyroid lesion (G), most of the primary tumors (H), and the surrounding immune cells (I). LTB was positive for tumor cells within a lymph node, as revealed by the brown immunostaining (I). Original magnification, 200 (A–D and I) and 400 (E–G and H).

Comparing a matched pair may help control against expression roles in a variety of fundamental biological processes, including changes that are unrelated to the metastatic process. cytoskeleton organization, cell lineage specification, and organ To understand PTC metastasis at a molecular level and identify development, and in oncogenesis (31). Thus far, there are no data potential prognostic markers and therapeutic target, we obtained on the functional role of LIMD2in metastasis. Although our SAGE expression profiles of a normal lymph node and from the same findings suggest that LIMD2is associated with metastases process, patient: a normal thyroid, primary tumor, and LNM. This is the first in vitro and in vivo analyses are necessary to test this hypothesis. report of gene expression profiling of matched samples in thyroid. Several observations made in this study may be assembled to The comparison of the mRNA from these four tissues revealed 31 reveal a new pathway associated with the LNM of PTC. First, our transcripts potentially associated with the metastasis. Eleven qPCR results showed an association between PTPRC (CD45) over- transcripts were tested by qPCR in a series of matched normal, expression and LNM. Immunohistochemistry confirmed that all primary tumor, and LNMs. LIMD2 and PTPRC were differentially LNMs express PTPRC, although its expression was found mainly in expressed between metastasis and primary tumor. LIMD2, PTPRC, the surrounding immune cells. One might suggest that these find- CD48, and LTB were significant between normal thyroid and ings may reflect immune modulation, rather than a role in the metastasis. The markers validated here were positive not only in the metastatic process. It has been suggested that inflammation asso- LNMs from classic PTC, but also in the metastases from the ciated with cancer development may be distinct from the normal follicular variant of PTC, independent of BRAF mutation status. inflammatory process on the basis of activation of the immune The present study shows, for the first time, that the expression of escape (32). Although the role of the adaptive immune response LIMD2 is associated with the metastatic process of PTC. LIMD2 in controlling the growth and recurrence of human tumors has derives its name from having two tandem copies of a LIM domain. been controversial, several studies have shown the sustaining In the LIM domain, there are seven conserved cysteine residues role of inflammatory mediators at distinct phases of malignant and a histidine sequence found in proteins that play important progression. It has been suggested that immune cells can release

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2007 American Association for Cancer Research. Predicted Markers for Lymph Node Metastasis inflammatory mediators for invasion, migration, and metastasis total thyroidectomy. Interference by antithyroglobulin antibodies (33), perhaps making the ‘‘soil fertile’’ for metastatic growth. in the blood is also present in f25% of patients. Circulating PTPRC is a prototype of the receptor-like protein tyrosine thyroid-specific transcripts such as TG, TPO, TSHR, NIS, and PDS phosphatase that has been previously shown to play a significant have been suggested as potential molecular markers of residual or role in CXCL12-induced and CXCR4-mediated chemotaxis through recurrent thyroid cancer (40–42). Conversely, several reports efficient coupling of CXCR4 with its signaling complex (34). It has suggested that these markers could not be used in the follow-up been shown that CXCR4 may also play a role in the outgrowth of of patients (43, 44). Therefore, there is much need for better the carcinoma metastases in lymph nodes by chemoattraction of markers. The new markers described here are candidates for the CXCR4-positive cells to the lymph nodes that highly express its possible use in conjunction with thyroglobulin or alone in the ligand CXCL12(35). Second, we have identified in the metastasis follow-up of patients with thyroid tumor recurrence. library an elevated expression of CXCR4. These results are in Although we focused on genes highly expressed in LNMs, these agreement with others where, in spite of tumor heterogeneity, most gene expression profiles may have other purposes. For example, primary tumor cells were CXCR4 negative, with only a few CXCR4- local invasion in the primary tumor likely precedes distant positive cells that were destined to metastasize (35, 36). Perhaps metastasis. Therefore, the primary tumor may already contain PTPRC plays a role in the outgrowth of carcinoma metastases in genes that predict poor prognosis. In this sense, a comparison done lymph nodes by facilitating the growth of CXCR4-expressing tumor among the matched normal and primary PTC would allow us to cells, but further investigation of this hypothesis is required. Third, identify genes highly expressed in primary tumors that could be an an additional indication that this signaling pathway may play a indication of host stromal response and poor prognosis. As crucial role in determining the metastatic phenotype is the fact expected, this analysis revealed similarities with microarray data that STAT5A, a member of the STAT family activated by CXCR4, done in primary thyroid tumors (16, 45, 46). For example, we was found highly expressed in our LNM library. Interestingly, it has identified and observed the previously reported transcripts highly been suggested that thyroid tumors with RET/PTC1 rearrangement, expressed in primary PTC, MET and S100A4. The validation a hallmark of PTC, use the CXCR4/CXCL12receptor-ligand analysis, however, proved that they are not associated with the late pathway to proliferate, survive, and migrate (33, 37). stages of the metastatic process. There were also other genes we implicated in PTC metastasis. In conclusion, our study identifies and validates a list of novel Although LTB mRNA expression was initially of borderline candidate markers associated with LNMs from PTC. Given the cri- statistical significance, we also looked at protein expression. tical role of tumor cell-host cell interactions in the metastatic process, Immunohistochemistry showed expression from the epithelial cells the genes identified here could be helpful to ascertain the molecular within the metastatic lesion in all metastases analyzed. Although basis of these cellular interactions. The genes and molecular pathways LTB was found expressed in three invasive and metastatic primary consistently associated with tumor invasion and metastasis could tumors, it was not expressed in normal thyroid and nonmetastatic also provide new targets for therapy. However, we should first PTC. One likely explanation is that LTB may be necessary for the determine if these biomarkers can improve patient care by earlier or early phases such as invasion as those required for the late phase of better detection of metastasis in patients with a diagnosis of PTC. this complex process. Interestingly, prevention of LTB-LTBR signaling is reported to inhibit tumor angiogenesis and neo- Acknowledgments vascularization, which results in tumor growth arrest (38). These Received 12/27/2006; revised 5/13/2007; accepted 6/14/2007. findings have prompted others to target the LTBR with agonist Grant support: NIH grant CA113461, the Virginia and D.K. Ludwig Fund for antibodies as a potential anticancer therapy (39). These results Cancer Research, and the Sa˜oPaulo State Research Foundation (FAPESP) from grants suggest that targeting the LTB signaling could also be a novel 04/15288-0 and 05/60330-8. J.M. Cerutti is investigator of the Brazilian Research Council (CNPq), G. Oler is a scholar from FAPESP, and G.J. Riggins is the recipient of approach to the treatment of metastatic PTC. the Irving J. Sherman M.D. Research Professorship. The project has been funded In addition to a better understanding of the metastatic process in part with federal funds from the National Cancer Institute, NIH, under contract in thyroid cancers, these markers have potential clinical use for N01-CO-12400. The costs of publication of this article were defrayed in part by the payment of page monitoring disease progression. Monitoring of serum thyroglobulin charges. This article must therefore be hereby marked advertisement in accordance (TG) levels has been used as gold-standard method in the follow-up with 18 U.S.C. Section 1734 solely to indicate this fact. The content of this publication does not necessarily reflect the views of policies of the of patients after total thyroidectomy and radioiodine therapy. Department of Health and Human Service, nor does mention of trade names, However, the value of this immunoassay is limited to patients with commercial products, or organizations imply endorsement by the U.S. Government.

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