Serpin Peptidase Inhibitor Clade a Member 1 As a Potential Marker for Malignancy in Insulinomas

Human Cancer Biology

Serpin Peptidase Inhibitor Clade A Member 1as a Potential Marker for Malignancy in Insulinomas Sandra Vale¤ riadeSa¤ ,1, 2 Maria Lu¤ cia Corre“ a-Giannella,1, 3 Ma¤ rcio Carlos Machado,3 Karin Krogh,4 Madson Queiroz de Almeida, 3 Maria AdelaideAlbergaria Pereira,3 4 4 1 Sheila Aparecida Coelho Siqueira, RoselyAntunes Patzina, FelI¤cia Satie Ibuki, Mari Cleide Sogayar,5 Marcel Cerqueira Ce¤ sar Machado,2 and Daniel Giannella-Neto1, 3

Abstract Purpose: The biological behavior of insulinomas cannot be predicted based on histopathologic criteria in which the diagnosis of malignancy is confirmed by the presence of metastases. In this study, microarray and quantitative real-time reverse transcription-PCR were applied to identify differentially expressed genes between malignant and nonmalignant insulinomas to search for useful biomarkers to recognize the metastatic potential of insulinomas. Experimental Design: CodeLink human bioarrays were used to analyze differences in f20,000 genes between six well-differentiated endocrine tumors of benign behavior compared with one well-differentiated endocrine carcinoma (WDEC) and three metastases of endocrine carcinomas (MEC). Quantitative real-time reverse transcription-PCR was used to validate differential expressions of five genes in a series of 35 sporadic insulinomas. Serpin peptidase inhibitor cladeA member1 (SERPINA1; a-1-antitrypsin) expression, identified as up-regulated in malignant insulinomas, was also evaluated by immunohistochemistry. Results: Analysis of microarray data resulted in 230 differentially expressed genes. Gene Ontology analysis identified serine-type endopeptidase activity and serine-type endopeptidase inhibitor activity as pathways presenting significant differential expression. Protease serine 2 and complement factor B (from serine-type endopeptidase activity pathway) were respectively confirmedasup-regulatedinwell-differentiatedendocrine tumors of benignbehavior (WDET) and inWDEC/MEC.Angiotensinogenand SERPINA1(fromserine-typeendopeptidaseinhibitoractivity pathway) were confirmed as up-regulated inWDEC/MEC. SERPINA1was shown to be expressed in 85.7% of malignant versus14.3% of nonmalignant insulinomas by immunohistochemistry. Conclusions: Our data are consistent to the possibility that SERPINA1is a marker of malignancy in insulinomas. Given the widespread availability of antibody anti-a-1-antitrypsin in pathology services, SERPINA1expression evaluation might be of clinical utility in recognizing patients more likely to develop an aggressive presentation.

Although insulinomas are rare endocrine tumors with an pancreatic endocrine tumors, the majority of insulinomas are estimated annual incidence of four cases per million popula- benign at the time of diagnosis (4). The average percentage of tion (1, 2), they are the most frequent functional islet of malignancy is 8.4% and its diagnosis can only be made in Langerhans cell tumors (3). In contrast to the other types of the presence of gross local invasion and/or distant metastases (5, 6). However, even the presence of metastasis cannot predict the clinical course, and there are patients with metastatic Authors’ Affiliations: 1Laboratory for Cellular and Molecular Endocrinology disease who present good clinical evolution. LIM-25, 2Division of Transplant and Liver Surgery, 3Division of Endocrinology, and Insulinomas with malignant behavior contain a higher 4Division of Pathology, University of Sao Paulo Medical School; and 5Department of number of genetic alterations than benign tumors, but only Biochemistry, Chemistry Institute, University of Sao Paulo, Sao Paulo, Brazil scarce information is available on molecular mechanisms Received 6/18/06; revised 4/19/07; accepted 5/17/07. Grant support: Fundac¸a‹ odeAmparoa' Pesquisa do Estado de Sa‹ oPaulogrants underlying insulinoma carcinogenesis. Accumulation of several 00/08448-0 and 02/01243-0. genetic alterations, including c-myc and transforming growth The costs of publication of this article were defrayed in part by the payment of page factor-a/epidermal growth factor receptor overexpression, c-K-ras advertisement charges. This article must therefore be hereby marked in accordance point mutations, and p53 protein overexpression, may con- with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research tribute to tumor development and/or progression from a Online (http://clincancerres.aacrjournals.org/). benign to a malignant phenotype (7, 8). Requests for reprints: Daniel Giannella-Neto, Laboratory for Cellular and The difficulty in predicting the biological behavior of Molecular Endocrinology (LIM-25), University of Sao Paulo Medical School, Av. insulinomas based on histopathologic criteria alone has long Dr. Arnaldo 455 #4307, 01246-903 Sa‹ o Paulo, Brazil. Phone: 55-11-3061-7253; Fax: 55-11-3061-7253; E-mail: [email protected]. been recognized (4). Jonkers et al. (9) have shown that F 2007 American Association for Cancer Research. chromosomal instability is a powerful indicator for the doi:10.1158/1078-0432.CCR-06-1477 development of metastatic disease in patients with sporadic

Clin Cancer Res 2007;13(18) September 15, 2007 5322 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. SERPINA1 Overexpression in Malignant Insulinomas insulinoma, and Vezzosi et al. (10) have suggested that the metastases of insulinomas and 30 primary tumors, 2 of them corres- presence of telomerase activity within a primary endocrine ponding to the primary insulinoma of hepatic metastases. tumor might indicate a malignant behavior. In the present study, Tumor fragments were resected by a single experienced surgeon, and microarray and quantitative real-time reverse transcription-PCR the fragments for RNA extraction were carefully removed from the central portion (by a single person) to avoid contamination with (RT-qPCR) analyses were applied in an attempt to identify normal pancreatic tissue. Most tumors were encapsulated and samples differentially expressed genes between malignant and nonma- evaluated in the microarray analysis presented an expansive growth lignant insulinomas with the aim of searching for useful bio- pattern, which reduces the possibility of acinar cell presence. markers to recognize the metastatic potential of insulinomas. Histologic and immunohistochemical analyses. Sections of all tumor tissues were histologically analyzed by H&E staining. Histologic examination included mitotic index (number of mitosis per 10 high- Materials and Methods power fields) and presence of perineural and vascular invasion. Immunohistochemical staining was done in sections of paraffin- Patients and tissue specimens. Tissue collection was carried out in embedded blocks by avidin-biotin peroxidase complex method using compliance to the Institution’s Ethics Committee and in accordance to antiserum anti-chromogranin A (BioGenex Laboratories), synaptophy- The Declaration of Helsinki, with informed and free consent being sin, insulin, proliferation-related Ki-67 antigen, and serpin peptidase required of each subject or subject’s guardian. From 1999 to 2005, inhibitor clade A member 1 (SERPINA1; a-1-antitrypsin; DakoCyto- tumor tissues were obtained from patients diagnosed with sporadic mation Denmark A/S). Mitotic index was graded from 0 to 3, perineural insulinomas based on the clinical features of Whipple’s triad of and vascular invasion was graded as 1 (no) or 2 (yes), proliferation symptoms with endogenous hyperinsulinemia. During laparotomy, rate (Ki-67) was graded as 0 (<2%) or 1 (>2%), and tumor sizes were tumor fragments were collected in sterile containers and immediately graded as 1 (V10 mm), 2 (>10 to V20 mm), 3 (>20 to V40 mm), or 4 frozen in liquid nitrogen. As shown in Table 1, 35 insulinoma samples (>40 mm). Based on these histopathologic features, insulinomas were collected from 33 patients were included in the study as follows: 5 liver classified according to the 2000 WHO International Classification of

Table 1. Clinical, histopathologic, and immunohistochemical features of tumor specimens studied

No. Tumor Outcome Follow-up Tumor size IMH IMH Ki-67 IMH SERPINA1 Mitotic index WHO patient type (mo) (mm) hormones (% cells) (% cells) (/10 HPF) classification

1 PT Remission 27 10 Ins <2 Negative 0 WDET-BB 2 PT Remission 56 8 Ins <2 Negative 1 WDET-BB 3 PT Remission 50 10 Ins <2 Negative 1 WDET-BB 4 PT Remission 41 17 Ins <2 Negative 0 WDET-BB 5 PT Remission 43 10 Ins <2 Negative 0 WDET-BB 6 PT Remission 42 13 Ins <2 Negative 0 WDET-BB 7 PT Recurrence 28 120 Ins/Gluc <2 >1 1 WDEC M Recurrence 28 * Ins — >1 — MEC 8 M Recurrence 71 15 Ins — Negative — MEC 9 M Deceased 27 * Ins — >1 — MEC 10 PT Remission 89 6 Ins <2 Negative 1 WDET-BB 11 PT Remission 36 8 Ins/SST <2 >1 1 WDET-BB 12 PT Remission 26 17 Ins <2 >1 1 WDET-BB 13 PT Remission 88 15 Ins <2 Negative 1 WDET-BB 14 PT Remission 89 10 Ins <2 Negative 1 WDET-BB 15 PT Remission 68 15 Ins <2 Negative 0 WDET-BB 16 PT Remission 25 9 Ins/SST <2 Negative NA WDET-BB 17 PT Remission 24 14 Ins <2 Negative 0 WDET-BB 18 PT Remission 85 7 Ins <2 Negative 1 WDET-BB 19 PT Remission 12 13 Ins <2 >1 1 WDET-BB 20 PT Remission 13 15 Ins <2 Negative 1 WDET-BB 21 PT Remission 34 25 Ins <2 Negative 0 WDET-UB 22 PT Remission 74 15 Ins/SST <2 Negative 2 WDET-UB 23 PT Remission 46 55 Ins/SST <2 Negative 2 WDET-UB 24 PT Remission 69 20 Ins >2 >1 0 WDET-UB 25 PT Remission 32 20 Ins/SST >2 Negative 0 WDET-UB 26 PT Remission 24 34 Ins >2 Negative 2 WDET-UB 27 PT Remission 43 20 Ins <2 Negative 0 WDET-UB 28 PT Remission 73 20 Ins <2 Negative 1 WDET-UB 29 PT Remission 52 15 Ins/SST >2 Negative 1 WDET-UB 30 PT Remission 24 26 Ins/Gluc NA Negative NA WDET-UB 31 PT Remission 22 18 Ins <2 Negative 0 WDET-UB 32 M Recurrence 15 c Ins — >1 — MEC 33 PT Deceased (sepsis) 62 20 Gluc >2 >1 1 WDEC M Deceased (sepsis) 62 10 Ins — >1 — MEC

Abbreviations: PT, primary tumor; M, metastasis; IMH, immunohistochemistry; Ins, insulin; Gluc, glucagon; SST, somatostatin; NA, not available; HPF, high-power field. *Laparotomic biopsies. cMultiple nodules (largest nodule, 20 mm in diameter).

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Endocrine Tumors as follows: well-differentiated endocrine tumors of Gene Ontology analysis. Microarray gene expression data were benign behavior (WDET-BB), confined to the pancreas, nonangioinva- analyzed by GeneSifter program.7 Gene ontology routine included in sive, no perineural invasion, <2 cm in diameter, less than two mitoses the program was chosen for further interpretation of the data about per 10 high-power fields, and <2% Ki-67–positive cells; well- molecular function-related genes according to Gene Ontology (GO) differentiated endocrine tumors of uncertain behavior (WDET-UB), Consortium categories (12).8 All the pathways in the GeneSifter confined to the pancreas and one or more of the following features: database were examined to determine whether a significant number z2 cm in diameter, 2 to 10 mitoses per 10 high-power fields, >2% of altered genes in GO term were affected. We examined z-scores that Ki-67–positive cells, angioinvasive, and perineural invasion; well- express the frequency of genes fulfilling an increase z2 in each GO term differentiated endocrine carcinoma (WDEC), in the presence of gross and compared with null hypothesis expected frequency for that GO local invasion and/or metastases; and metastasis of endocrine term based on the total number of genes examined on the array. The carcinoma (MEC; ref. 6). A single pathologist has evaluated all samples. z-score was derived by dividing the difference between the observed Study design. As a screening strategy to determine which genes were number of genes meeting the criterion in a specific GO term and the differentially expressed in malignant (WDEC and MEC) compared with expected number of genes based on the total number of genes in the nonmalignant insulinomas (WDET-BB and WDET-UB), we did gene array meeting the criterion. This value was then divided by the SD of expression profiling by microarray analysis of six tumors classified as the observed number of genes under a hypergeometric distribution. WDET-BB compared with four malignant tumors, one classified as Positive z-scores indicate GO terms with a greater number of genes WDEC and three as MEC. The criteria for discovering differentially meeting the criterion than is expected by chance. Negative z-scores expressed genes by microarray analysis are comprehensively described indicate GO terms with fewer genes meeting the criterion than expected in Microarray Data Analysis section. To validate microarray results, five by chance. A z-score near zero indicates that the number of genes differentially expressed genes were selected and their expressions were meeting the criterion approximates the expected number. A z-score of relatively quantified by RT-qPCR in a larger set of insulinoma samples >2 is considered a statistically significant association (approximately as follows: WDET-BB (n = 17), WDET-UB (n = 11), WDEC (n = 2), equivalent to a P V 0.05) between the differentially regulated genes and and MEC (n = 5). SERPINA1 (a-1-antitrypsin), identified as up- their corresponding GO terms (13, 14). A false discovery rate of 5% was regulated in malignant insulinomas (WDEC/MEC), was also evaluated also set as a cutoff for statistical significance. by immunohistochemistry in all studied tumor tissues. Quantitative real-time reverse transcription-PCR. Five genes identi- Microarray. Gene expression profiling was done using CodeLink fied by microarray analysis were also evaluated by relatively RT-qPCR UniSet Human 20K I Bioarray (GE Healthcare Biosciences). The to validate transcriptional responses in a large tumor series: protease Minimum Information about a Microarray Experiment checklist serine 2 (PRSS2), chymotrypsinogen B1 (CTRB1), SERPINA1, angiotensi- containing all information is included as Supplementary Data 1. nogen (AGT), and complement factor B (CFB). Relative expression of Furthermore, a detailed description of the microarray experiment, each gene was measured in reference to the housekeeping gene according to the Minimum Information about a Microarray Experiment proteasome 26S ATPase subunit 6 (PSMC6) and normalized with a criteria, is also available online at Gene Expression Omnibus (accession reference sample from a unique insulinoma patient. number GPL2891).6 For stability comparison of some candidate housekeeping gene Total RNA from tumor tissues was prepared using Trizol reagent presented in CodeLink array, the NormFinder software (15) was (Invitrogen) according to standard protocols provided by the manu- applied (freely available online).9 It is a Microsoft Excel add-in that facturer. RNA samples were purified with RNeasy Mini kit (Qiagen calculates the stability values of individual candidate reference genes for GmbH), and total RNA integrity was assessed by denaturing agarose gel normalization. Stability value is based on combined estimate of electrophoresis. Concentrations were quantified by spectrophotometry intragroup and intergroup expression variations of the genes studied. (GeneQuant Pro DNA/RNA Calculator, GE Healthcare Biosciences), The PSMC6, used as a reference gene in a previous study (16), presented and 2 Ag were used from each studied subject. Target synthesis and one of the lowest stability value compared with well-known house- hybridizations were done with the CodeLink Expression Assay kit (GE keeping genes. Healthcare Biosciences) according to the manufacturer’s instructions. Total RNA was extracted from 35 insulinomas tissues as mentioned Microarray data analysis. The f20,000-spot intensities on the previously in microarray analysis. Reactions were conducted on a Rotor- microarray image were quantified in CodeLink Expression Array Gene RG-3000 (Corbett Research) using SuperScript III Platinum SYBR Software (GE Healthcare Biosciences), filtering out genes with missing Green One-Step qRT-PCR kit (Invitrogen). RT-qPCR was done in a final spots, and the expression values from duplicated were averaged. volume reaction of 25 AL with 5.0 AL RNA sample (20 ng/AL), 0.5 AL Intensity of each spot was divided by the intensity median of the array SuperScript III RT/Platinum Taq Mix, 12.5 AL2Â SYBR Green Reaction to provide a scaled and comparable number across multiple arrays. Mix (containing 3 mmol/L MgSO4), and 0.2 sense/antisense primer. The expression values of these genes were normalized individually Negative samples were run for each RT-qPCR assay consisting of no across all arrays. Expression ratio was calculated by log2 between six RNA in the reverse transcriptase reaction and no cDNA in the PCR. To WDET-BB and four WDEC/MEC. Unpaired Student’s t test was used confirm accuracy and reproducibility of RT-qPCR, the intra-assay for two-group statistical comparisons. Differentially expressed genes coefficients of variation for each gene calculated with five replicates of were selected according three basic criteria: presence of at least 40% of a unique sample within one run varied from 3.6% to 8.3% and the ‘‘G’’ (good) flag in analyzed arrays, differential expression either interassay coefficients of variation for each gene calculated with the very up-regulated or down-regulated by at least 2-fold in the average same sample repeated in duplicate in each assay varied from 6.1% to WDET-BB compared with WDEC/MEC expression data, and statisti- 9.8%. Reactions were carried out under the following cycling cally significant log2 ratio at probability levels of P < 0.05. Correction conditions: 5 min at 50jC and 5 min at 95jC followed by 35 three- for multiple testing was then done using the method of Reiner et al. temperature cycles (20 s at 95jC, 30 s at 55jC, and 30 s at 72jC). (11) to derive a false discovery rate estimate from the raw P values. Fluorescence changes were monitored after each cycle, and melting A false discovery rate of 5% was set as a cutoff for statistical curve analysis was done at the end of cycles to verify PCR product significance. Supervised hierarchical clustering analysis was done identity (72jC ramping to 99jCat0.2jC/s with continuous fluores- among differentially expressed genes and different tumor tissue cence readings). Specificity of amplicons was also ensured by agarose analyzed. All statistical analysis was done using JMP Release 5.1.1 software (SAS Institute, Inc.). 7 http://www.genesifter.net/web/ 8 http://www.geneontology.org/GO.doc.html 6 http://www.ncbi.nlm.nih.gov/geo/ 9 http://www.mdl.dk

Clin Cancer Res 2007;13(18) September 15, 2007 5324 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. SERPINA1 Overexpression in Malignant Insulinomas gel electrophoresis to visualize a unique product fragment with appropriate size. Gene-specific primer pairs were located on two adjacent exons to achieve a high level of specificity and to avoid detection of genomic DNA. Primers were designed to have similar guanosine-cytosine content and melting temperatures by using Primer3 Program10 (17): PRSS2 (NM_002770), 5¶-CCCTACCAGGTGTCCTT- GAA-3¶ (sense) and 5¶-AGTCCGGCTGTTGTATTTGG-3¶ (antisense; 204-bp product); SERPINA1 (NM_000295), 5¶-CACCCACGATATCAT- CACCA-3¶ (sense) and 5¶-TCAGTCCCTTTCTCGTCGAT-3¶ (antisense; 237-bp product); AGT (NM_000029), 5¶-GAACTGGATGTTGC- TGCTGA-3¶ (sense) and 5¶-GCTGTTGTCCACCCAGAACT-3¶ (antisense; 189-bp product); CFB (NM_001710), 5¶-TAATGCAGACTGGGT- CACGA-3¶ (sense) and 5¶-ATAGCAAGTCCCGGATCTCA-3¶ (antisense; 239-bp product); CTRB1 (NM_001906), 5¶-AGGAGGATCACC- GACGTG-3¶ (sense) and 5¶-TGGAGGTGGAGCAGGTGT-3¶ (antisense; 151-bp product); and PSMC6 (NM_002806), 5¶-GCTGCGTCCAGGAA- GATTAG-3¶ (sense) and 5¶-TGCGAACATACCTGCTTCAG-3¶ (antisense; 196-bp product). To evaluate the amplification efficiency of each target and housekeeping genes, standard curves were constructed from a reference sample of RNA using duplicate serial dilutions with six different RNA concentrations (0.2, 0.8, 3.1, 12.5, 50, and 200 ng/AL). The mathematical model described by Pfaffl (18) was used to evaluate the relative expression ratio for all genes compared with PSMC6 and efficiency values for each set of primers, additionally normalized to the amount of RNA reference. Data were analyzed by an unpaired, two- tailed Student’s t test with statistical significance attributed to P < 0.05. Statistical analysis of RT-qPCR. Statistical tests were evaluated by JMP Version 5.1 statistical computer program (SAS Institute). Because assumptions for a parametric test were not valid (P < 0.05, Kolmogorov-Smirnov), all data were evaluated by Kruskal-Wallis ANOVA complemented by Scheffe` test as a multiple comparison method. Statistical power was calculated for each comparison and considered adequate when >80%. The Spearman test was used to assess the statistical significance of correlations between mRNA expression and histopathologic data that could imply any malignant feature, such as mitotic index, perineural and vascular invasion, proliferation rate (Ki-67), and tumor size. These variables were graded as described in the Histologic and Immunohistochemical Analyses section. Data are expressed in mean F SE. Statistical significance was fixed at probability levels of <0.05.

Results

Clinical and histopathologic findings. The cohort observed in our series was similar to other large series of patients (1) with hypoglycemia syndrome. Disease duration ranged widely from 1 to 168 months with a median of 24 months. The frequency of malignancy was 15%. Clinical and biochemical data are summarized as Supplementary Table S1. Table 1 describes outcome data and follow-up for all patients and relevant histologic and immunohistochemical features from 35 tumor specimens analyzed in 33 patients. Among the patients with WDET-BB (median follow-up, 42; range, 12-89 months) and WDET-UB (median follow-up, 43; range, 22-74 months), no recurrence of disease was observed at any time during follow-up. Mean largest diameter of the tumors was 19.7 F 20.5 mm (mean F SD) ranging from 6 to 120 mm. There was no evidence of perineural invasion and necrosis in any samples. Few tumors (8.6%) presented mitotic index more than two mitoses per 10 high-power fields. None of the tumors analyzed presented necrosis in the histopathologic Fig. 1. Expression profiles of 10 samples representing 6 WDET-BB and 4 WDEC/ MEC analyzed by supervised hierarchical clustering using 230 differentially 10 http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi expressed genes. Row, single gene; column, tissue sample.

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Table 2. GO Consortium categories unveiled by GeneSifter analysis of microarray data obtained in six WDET-BB in comparison with four WDEC/MEC

GO ID Molecular function Totals Z-score List Up Down Array Up Down

5488 Binding 511 252 259 7,610 -0.71 0.5 3824 Catalytic activity 254 134 120 3,793 0.62 -0.8 16787 >Hydrolase activity 112 54 58 1,552 0.21 0.92 8233 >>Peptidase activity 32 16 16 445 0.25 0.29 4175 >>>Endopeptidase activity 25 16 9 311 1.74 -0.45 4252 >>>>Serine-type endopeptidase activity 14 11 3 120 3.52 -0.52 4871 Signal transducer activity 118 73 45 1,871 1.35 -2.51 5215 Transporter activity 80 50 30 1,181 1.71 -1.64 30528 Transcription regulator activity 58 24 34 993 -1.78 0.14 5198 Structural molecule activity 44 22 22 532 0.98 1.03 30234 Enzyme regulator activity 42 28 14 535 2.42 -0.97 4857 >Enzyme inhibitor activity 21 15 6 187 3.53 -0.11 30414 >>Protease inhibitor activity 15 13 2 98 5.43 -0.72 4866 >>>Endopeptidase inhibitor activity 15 13 2 97 5.48 -0.71 4867 >>>>Serine-type endopeptidase inhibitor activity 8 6 2 62 2.75 -0.05 5554 Molecular function unknown 32 13 19 565 -1.47 0.02 3774 Motor activity 7 4 3 113 0.09 -0.41 45182 Translation regulator activity 4 2 2 85 -0.53 -0.51 30188 Chaperone regulator activity 2 0 2 6 -0.46 4.08 16209 Antioxidant activity 1 0 1 34 -1.09 -0.13 45735 Nutrient reservoir activity 0 0 0 1 -0.19 -0.19

NOTE: Genes changed refers to the number of distinct genes that met the criteria considered for the expression data set analysis. The table shows molecular function GO IDs and GO terms, number of perturbed genes (up-regulated and down-regulated in relation to WDET-BB), and z-scores for up-regulated and down-regulated genes. Serine-type endopeptidase activity and serine-type endopeptidase inhibitor activity pathways presented a z-score >2 for genes up-regulated in WDET-BB. Z-score is the standard statistical test under the hypergeometric distribution used to check if the genes in the GO term would show a difference in the ratio of genes meeting the criteria considered for the expression data set analysis compared with the background percentage in the entire GO. A positive z-score indicates that there are more genes meeting the criteria in the GO term than would be expected by random chance. A negative z-score indicates that the there are fewer genes meeting the criteria than would be expected by random chance.

examination. Immunohistochemical study for Ki-67 exhibited depicting the relationship between gene expression and tissue an important immunoreaction (>2% of positive cells) in 14.3% types, according to the histologic classification. A complete of all analyzed tumor tissues. Ki-67 immunostaining and listing of transcriptional changes from all differentially expressed mitotic index were not applicable for analysis in metastatic genes is provided as Supplementary Table S2. tumors. Immunoperoxidase staining for chromogranin A and Biological pathway significance. GO analysis used to map synaptophysin was positive in all tumor tissues analyzed. differentially expressed genes to candidate biological terms Hormonal immunohistochemical analysis showed positive identified two pathways with significant z-score (positive and insulin staining in all samples tissues, except in the primary >2) correlated to molecular function GO terms, namely tumor from sample 33, despite unequivocal clinical hypogly- chaperone regulator activity (GO ID: 30188; z-score, 4.08) cemia. Immunoreaction for other hormones was observed and enzyme regulator activity (GO ID: 30234; z-score, 2.42), in nine tissue samples: six positive somatostatin staining (two respectively, more disturbed in WDET-BB and WDEC/MEC. WDET-BB and four WDET-UB) and three positive glucagon Although significant change has been observed in chaperone immunoreaction (one WDET-UB and two WDEC). regulator activity, this pathway was not chosen because only six Identification of genes differentially expressed by microarray. genes were represented on the array from which two were Six WDET-BB (patients 1, 2, 3, 4, 5, and 6) and four WDEC/ differentially expressed. According to previous reports, GO MEC (patients 7PT, 7M, 8, and 9) were analyzed for gene terms presenting fewer than five genes changed have not been expression using CodeLink UniSet Human 20K I Bioarray considered for validation (15, 16). On the other hand, in the containing f20,000 known human genes. After data normal- enzyme regulator activity (GO ID: 30234) pathway, 42 from ization, 13,060 genes presented reliable measurements of at 535 genes were altered. A group of genes with related functions least 40% of G flag value as assigned by the CodeLink software. represented by the final branch of this GO term is the serine- Student’s t test analysis of the individual gene expression ratios type endopeptidase inhibitor activity pathway (GO ID: 4867; (log2 ratio between WDET-BB and WDEC/MEC) resulted in z-score, 2.75) containing eight genes differentially expressed 230 considered differentially expressed, defined as those (two up-regulated in WDET-BB and six up-regulated in WDEC/ meeting criteria mentioned above. From them, 120 genes were MEC). Another closely related pathway, serine-type endopep- overexpressed in WDET-BB and 110 in WDEC/MEC. Supervised tidase activity (GO ID: 4252), also presented a relevant z-score hierarchical clustering done on the log2 values for the 230 genes value, although its initial molecular function GO term (catalytic differentially expressed resulted on the dendrogram (Fig. 1), activity, GO ID: 3824) did not had a z-score >2.0. Table 2

Clin Cancer Res 2007;13(18) September 15, 2007 5326 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. SERPINA1 Overexpression in Malignant Insulinomas depicts all GO terms from molecular function and the final mRNA levels (>2-fold) of four of five genes were concordant branches of serine-type endopeptidase activity and serine-type with those observed in microarray analysis. As shown in Fig. 2A, endopeptidase inhibitor activity pathways. mRNA expression of PRSS2 presented increased expression Gene expression by RT-qPCR. Among the up-regulated genes in the group of WDET (BB and UB; 0.87 F 0.40) compared in WDET-BB (Supplementary Table S2) belonging to serine- with malignant tumors (WDEC and MEC; 0.001 F 0.001). type endopeptidase activity pathway, we selected for RT-qPCR The opposite regulation was observed in mRNA content of validation the two most differentially expressed genes, CTRB1 SERPINA1 (Fig. 2B), AGT (Fig. 2C), and CFB (Fig. 2D), with and PRSS2, and with 7.9- and 5.2-fold changes, respectively. increased expression in malignant insulinomas (WDEC and Similarly, along with the up-regulated genes in WDEC/MEC MEC; 76.00 F 39.25, 193.46 F 127.86, and 20.43 F 16.34, (Supplementary Table S2) from serine-type endopeptidase respectively) than in WDET (BB and UB; 0.47 F 0.14, 2.66 F inhibitor activity pathway, the genes AGT (6.1-fold change) 1.08, and 0.75 F 0.27, respectively). Comparison between and SERPINA1 (6.6-fold change) were chosen. CFB gene, WDET-BB and WDET-UB for all studied genes did not present belonging to serine-type endopeptidase activity pathway and any statistical difference. No statistically significant difference found to be up-regulated in WDEC/MEC (3.9-fold change), was was observed in CTRB1 expression between the different also selected for validation because its expression had been histologic groups [WDET-BB and WDET-UB (1.64 F 0.83) previously identified in other human cancer cells (19–21). and WDEC and MEC (0.02 F 0.01)] as depicted in Fig. 2E. Transcriptional changes of PRSS2, CTRB1, SERPINA1, AGT, Tumors identified as plurihormonal by immunohistochem- and CFB were examined by RT-qPCR using a set of independent ical staining showed the same average mRNA expression as RNA samples prepared from 35 tumor tissues. Changes in other tumor types in all genes analyzed.

Fig. 2. mRNA expression values of PRSS2 (A), SERPINA1 (B), AGT (C), CFB (D), and CTRB1 (E) in sample tumors classified according toWHO classification (WDET-BB, WDET-UB,WDEC, and MEC). Box diagram comparing relative mRNA expression levels of genes in WDET-BB/WDET-UB compared with WDEC/MEC. The horizontal line within the box plot represents the median value, the box plot limits refer to 25th to 75th percentiles, and the box plot bars include the 10th to 90th percentiles for mRNA levels.

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No statistical significant correlation was found between between benign and malignant insulinomas following sup- mRNA expression of studied genes and histopathologic pressive subtractive hybridization (22). Among the genes variables by Spearman rank test (data not shown). identified as up-regulated in WDEC/MEC, one gene from Immunohistochemical analysis of SERPINA1. Six of 7 serine-type endopeptidase activity pathway, CFB, and two (85.7%) samples of malignant insulinomas (including WDEC genes belonging to serine-type endopeptidase inhibitor activity and MEC) showed positive immunohistochemical staining pathway, SERPINA1 (also known as a-1-antitrypsin) and AGT for SERPINA1, whereas only 4 of 28 (14.3%) WDET samples (also known as SERPINA8), were chosen for RT-qPCR presented positive immunostaining for SERPINA1 (Fig. 3). validation. PRSS2, CFB, SERPINA1, and AGT had their From the positive cases, three corresponded to WDET-BB differential expressions confirmed between WDET-BB and (patients 11, 12, and 19) and one to WDET-UB (patient 24). WDEC/MEC in 35 tumor samples. Trypsinogen-2 is the precursor of PRSS2, which is produced in Discussion pancreatic acini (23) and also by tumors (named tumor- associated trypsinogen 2; ref. 24, 25), whose expression has been In the present study, we compared global gene expression shown to correlate with the malignant phenotype of profiles between WDET-BB and WDEC/MEC using microarray some cancer cells through activation of latent matrix metallo- analysis in an attempt to identify molecular markers for an proteinases, which may stimulate tumor invasion and metas- aggressive biological behavior in insulinomas. Even recognizing tasis (24, 26, 27). the limitation of WHO classification in predicting malignancy, PRSS2 expression in insulinomas might reflect either the this categorization was used because it constitutes a standard- presence of acinar cells in the tumor or production of ized pathologic evaluation that has been considered useful for trypsinogen-2 by the tumoral cells. Although in the current clinical purposes. Despite the small number of tumors initially study immunohistochemistry for specific acinar cell markers evaluated, the microarray findings seem to be representative had not been done, a carefully histologic analysis did not show based on the concordant results of four (PRSS2, CFB, SERPINA1, morphologic features of acinar cell differentiation, characteriz- and AGT) of five selected genes validated by RT-qPCR on 35 ing the tumors as purely endocrine. Yantiss et al. (28) have fresh-frozen tissue specimens. recently studied pure pancreatic endocrine tumors, including From f20,000 transcripts analyzed, 230 genes showed insulinomas, for the presence and degree of acinar differenti- >2-fold differential expression between tumor classes. Our ation by immunohistochemical analysis. Overall, 66% of objective to search for markers that could be validated by tumors stained positively for at least one acinar cell marker, RT-qPCR was based on stringent selection criteria and probably 37% for trypsin, and 25% for chymotrypsin. The authors did neglected several subtle modifications in gene expression that not find any correlation between the expression of acinar cell will be considered for future studies. The supervised hierarchi- markers and clinical outcome and suggest that cytochemical cal clustering of these 230 genes resulted into two distinct evidence of concomitant acinar and endocrine differentiation tumor groups (WDET-BB and WDEC/MEC), represented by strongly suggests the existence of a common progenitor stem nodes 1 and 2 (Fig. 1). cell. Based on this premise, we speculate that, about insulino- Two genes belonging to serine-type endopeptidase activity mas, a higher degree of differentiation would explain the higher pathway, CTRB1 and PRSS2, were selected for validation by expression of the acinar cell marker PRSS2 in well-differentiated RT-qPCR based on the magnitude of their expression ratio tumors. Alternatively, we cannot rule out that lower expression differences as well as on our interest in studying PRSS2, which of this gene in WDEC/MEC is associated with promoter was previously identified as one differentially expressed gene hypermethylation as already reported by Yamashita et al. (29)

Fig. 3. Representative immunohistochemistry of WDET-BB (A)andWDEC(B) presenting negative and positive immunostaining, respectively, with anti-SERPINA1antibody. Magnification, Â40.

Clin Cancer Res 2007;13(18) September 15, 2007 5328 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. SERPINA1 Overexpression in Malignant Insulinomas in esophageal squamous cell carcinomas and gastric adenocar- The expression of SERPINA1, which codes for a-1-antitrypsin, cinomas. a major plasma serpin that has a broad inhibitory spectrum CFB, the zymogen of the catalytic site of the C3/C5 against serine proteases, has been related to the invasive convertase of the alternative pathway of complement (30), potential of gastric (34), lung (35), and colorectal adenocarci- had its up-regulation in WDEC/MEC validated by RT-qPCR. nomas (36). Our finding of SERPINA1 mRNA up-regulation CFB expression has already been reported in the exocrine in WDEC/MEC agrees with data published by Hansel et al. (37) pancreas, where it is regulated by interleukin-1b, tumor necrosis reporting SERPINA1 overexpression in metastatic versus non- factor-a,andIFN-g and probably synthesized by ductal metastatic tumors in a study that evaluated global gene epithelial cells (19), as well as in human cancer cells, including expression profiles from well-differentiated pancreatic endo- gastric (20) and astrocytoma cell lines (31). Doustjalali et al. crine neoplasms. Although SERPINA1 expression had not been (21) have recently shown immunoreactivity for CFB in validated in this series, which differs from ours in that is not malignant breast lesions, whereas benign lesions negatively composed only of insulinomas, its identification as up-regu- stained for this protein. Whether expression of CFB lated in metastatic tumors also suggests that SERPINA1 plays a is a direct determinant of tumor aggressiveness or it is a role in pancreatic endocrine neoplasia progression. The partici- consequence of host defense mechanisms as the tumor becomes pation of this protein in tumoral invasiveness seems to be more invasive remains to be elucidated. related to its degradation by matrix metalloproteinases, Another gene identified as up-regulated in WDEC/MEC resulting in the production of a COOH-terminal fragment that was AGT, previously described in human insulinomas, where increases tumor growth in vivo, possibly due to its modulatory the RNA abundance was shown to be higher than in normal effects on natural killer cell activity against the tumor cells (38). pancreas (32). AGT, the product of this gene, is cleaved by Immunohistochemical analysis identified SERPINA1 expres- renin to generate angiotensin I, which is subsequently sion in 85.7% of malignant versus 14.3% of nonmalignant converted into angiotensin II by angiotensin I–converting insulinomas. Interestingly, the MEC (sample 8) presenting enzyme. AGT shares structural homologies with the serine negative immunostaining for SERPINA1 was a hepatic metas- protease inhibitor (serpin) family of proteins, but it has no tasis that determined hypoglycemia 12 years after the resection inhibitory activity. Ce´le´rier et al. have shown that AGT, as of the primary tumor, characterizing an indolent clinical course well as the fragment resulting from its cleavage (des[angio- in spite of being a metastatic tumor. The four patients whose tensin I]AGT), presents antiangiogenic activity. Because tumors classified as WDET-BB or WDET-UB presented positive angiogenic effect is associated to angiotensin II, they SERPINA1 immunostaining are being closely followed to assess suggested that the final effect on angiogenesis depends on their long-term evolution. local conditions, such as the clearance rate of angiotensin II To our knowledge, this is the first study on a series and the presence or absence of renin (33). Considering that comprising only insulinomas approached by microarray renin,aswellasangiotensin I–converting enzyme RNA analysis that provided the identification of genes associated expression, was detected in all tumor sample evaluated by with tumoral malignancy. Considering the widespread avail- microarray (data not shown), it is tempting to speculate that ability of antibody anti-a-1-antitrypsin in pathology services, up-regulation of AGT in WDEC/MEC is associated with a the confirmation of SERPINA1 overexpression in larger series final proangiogenic effect, which contributes to tumor of WDEC might be of clinical utility in identifying patients growth. more likely to develop aggressive disease.

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Sandra Valéria de Sá, Maria Lúcia Corrêa-Giannella, Márcio Carlos Machado, et al.

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