Topographic Molecular Profile of Pheochromocytomas: Role of Somatic Down-regulation of Mismatch Repair Patients and Methods Case Selection

Context and Objective: Despite extensive molecular investigation of adrenal pheochromocytomas, no information is available on their molecular and mismatch repair (MMR) profiles by topographic compartments. Design and Setting: Microdissected samples from the peripheral and internal zones of 143 pheochromocytomas from a referral hospital (95 sporadic and 48 associated with multiple endocrine neoplasia type 2A) were selected for loss of heterozygosity and single nucleotide polymorphism analyses. Five polymorphic DNA regions from TP53, RB1, WT1, and NF1 were systematically studied by PCR-denaturing gradient gel electrophoresis. Patients, Outcome Measures, and Interventions: Pheochromo-cytomas were classified as malignant (16 sporadic tumors with distant metastases), locally invasive (30 sporadic tumors showing ret-roperitoneal infiltration only), and benign (all remaining tumors). Statistical differences were evaluated using Fisher's exact test. MMR was assessed by MLH1/MSH2 sequencing and immunostaining in pheochromocytomas with two or more abnormal microsatellites. No interventions were performed in this study. Results: Loss of heterozygosity/single nucleotide polymorphism in-More genetic abnormalities involving the peripheral compartment were revealed in 34 pheochromocytomas (23.8%): 12 of 16 malignant, 10 of 30 locally invasive, and 12 of 97 benign. Multiple and coexistent genetic abnormalities characterized malignant pheochromocytomas (P Ͻ 0.001), whereas locally invasive pheochromocytomas showed a significantly higher incidence of NF1 alterations (P Ͻ 0.001). No mutations were identified in MLH1/MSH2, but MMR proteins significantly decreased in peripheral compartments. Conclusions: Multiple microsatellite alterations and topographic intratumor heterogeneity characterize malignant pheochromocyto-mas, suggesting a multistep tumorigenesis through somatic topo-graphic down-regulation of MMR proteins. Locally invasive pheo-chromocytomas reveal topographic heterogeneity and single-locus microsatellite alterations, especially involving NF1. P HEOCHROMOCYTOMAS (PCC) ARE heterogeneous tumors (1– 4) that show clonal expansions due to loss of heterozygosity (LOH) of several tumor suppressor genes (TSG) located on chromosomes 1p, 3p, 17p, and 22q (5–9). Those markers have revealed significant association with clinicopathological parameters, such as tumor volume (5), or distinctive transformation pathways (7), although their relative incidence is quite variable because of the limited number of cases analyzed. A monoclonal tumor origin is supported by concordant TSG abnormalities, such as point mutations or single nucleotide polymorphisms (SNPs) (10, 11), which have been found associated with LOH of certain loci (12). The coexistence of several genetic abnormalities and intratumor heterogeneity would be the expression of either tumor cell selection or a simple passive byproduct of genetic instability (10, 13, 14). However, the association of multiple genetic alterations would become statistically less probable as the number of molecular markers increases (10, …

A monoclonal tumor origin is supported by concordant TSG abnormalities, such as point mutations or single nucleotide polymorphisms (SNPs) (10,11), which have been found associated with LOH of certain loci (12).The coexistence of several genetic abnormalities and intratumor heterogeneity would be the expression of either tumor cell selection or a simple passive byproduct of genetic instability (10,13,14).However, the association of multiple genetic alterations would become statistically less probable as the number of molecular markers increases (10,11,13,15) and is useful to test clonal expansions in tumors (10,11).Although genetic abnormalities are probably asymmetrically acquired (16), there is a correlation with tumor cell topography, as demonstrated in bladder and colon (13); the topography role has not been studied in the adrenal gland.Any potential topographic segregation of tumor cells will influence interpretations of the results and would help in designing more effective therapies to maximize the effect in the most sensitive areas (e.g.zones with higher proliferation).
No information is available on the molecular and mismatch repair (MMR) profiles of PCC by topographic compartments.This study investigates the TSG microsatellite pattern in sporadic and multiple endocrine neoplasia (MEN) 2A-associated PCC, using microdissected samples from the peripheral and internal compartments to assess both LOH and somatic SNP of TSG controlling G 1 -S transition (TP53, RB1), RAS pathway (NF1), and development (WT1).

Case selection
Sporadic (n ϭ 95) and MEN 2A (n ϭ 48) PCC were included in this study.MEN 2A patients revealed adrenal medullary hyperplasia (17), but only nodules larger than 1 cm were included in this study (3).
Standard protocols were followed for PCC sectioning and sampling

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(at least one block per centimeter of tumor), being appropriate archival material available in all cases.The same areas in consecutive sections were used in each study, and their cellular composition was confirmed in adjacent hematoxylin-eosin-stained sections.This protocol was approved by the Hospital Research Board and ethical committee and complied with their requirements.

TSG microsatellite analysis
DNA was extracted from the most cellular areas of peripheral and internal compartments after microdissecting at least 100 cells (ϳ0.4 mm 2 ) from two 20-m unstained paraffin sections/compartment (Fig. 1).Appropriate controls were included for each test (adrenal medulla, adrenal cortex, and periadrenal soft tissue).
Interpretation and inclusion criteria in each sample were previously reported (11,13,(21)(22)(23).Allelic imbalance was densitometrically evaluated (EC model 910 optical densitometer, EC Apparatus Corp., St. Petersburg, FL), considering evidence of only LOH allele ratios of 4:1 or more in any TSG; otherwise, retention of heterozygosity (ROH) was assigned (13,22).This ratio represents 80% of clonal cells in the sample and was used to increase the detection specificity (10,11,23).Additional allele bands present in tumor samples, but not in the corresponding controls, were considered evidence of somatic SNP by PCR/denaturing gradient gel electrophoresis (DGGE) (13).

DNA sequencing
All extra bands were cut from gels, and DNA was purified using a QIAquick gel extraction kit (QIAGEN, Valencia, CA).The amplified product was diluted 20-fold in Tris-EDTA buffer, and 1 l of the diluted reaction product was subjected to a second round of PCR amplification using the appropriate primers for 30 cycles under the above conditions.Normal and extra bands from tumor-derived samples were PCR amplified along with the corresponding controls using a high-fidelity polymerase, Platinum PFX (Invitrogen Life Technologies, Inc., Carlsbad, CA).PCR products were directly sequenced after purification (QIAquick PCR purification kit, QIAGEN).All sequencing was performed on an ABI PRISM 3700 automated DNA analyzer (Applied Biosystems, Foster City, CA), and the sequence data were analyzed using the program Sequencher (Gene Codes Corp., Ann Arbor, MI), which reverses and complements the antisense strand.All mutations were confirmed by sequencing in both directions and are indicated by an N in the sequencing chromatogram.
MLH1/MSH2 exons were completely sequenced in cases with microsatellite abnormalities in at least 40% loci and/or complete loss of mlh1/msh2 immunoreactivity as well as in a representative sample from mlh1/msh2-immunoreactive cases (30 PCC), which was used as the control group.

Immunohistochemical expression of mlh1 and msh2
The sections were mounted on positively charged slides (SuperFrost Plus, Fisher Scientific, Fair Lawn, NJ), baked at 60 C for 2 h, and processed as previously described (13,20,24).After routine dewaxing and rehydration, endogenous peroxidase quenching, and antigens heat retrieval, the slides were transferred to a moist chamber.Nonspecific binding was blocked with polyclonal horse serum, and sections were incubated with monoclonal primary antibodies (overnight, 4 C): 2 g/ml for hMLH1 (clones G168 728 and G168-15, BD Pharmingen, San Diego, CA) and hMSH2 (clone FE11, Oncogene Research Products, San Diego, CA).Then sections were serially incubated with biotinylated antimouse antibody and peroxidase-labeled avidin-biotin complex.The reaction was developed under microscopic control, using 3,3Ј-diaminobenzidine tetrahydrochloride with 0.3% H 2 O 2 as chromogen (Sigma-Aldrich Corp., St. Louis, MO), and the sections counterstained with hematoxylin.Both positive (reactive lymph node) and negative (omitting the primary antibody) controls were simultaneously run.

Quantification of positive nuclei
At least 50 high-power fields (7.6 mm 2 ) were screened in each pathological group, beginning in the most cellular area.The number of positive nuclei was expressed per high-power field and per 1000 tumor cells, and the average and sd were calculated in each pathological condition and patient as previously described (25,26).The positivity threshold was experimentally established at the positive control in each staining batch.Only nuclei with staining features similar to those of their corresponding positive control were considered positive for any marker.

Statistical analysis
The results were compared by tumor compartment (peripheral vs. internal) in PCC classified by the presence or absence of genetic heterogeneity, the genetic background (sporadic vs. MEN 2A), and the biological behavior (nonmetastatic vs. metastatic).Qualitative variables were compared using Fisher's exact tests, and quantitative variables were compared by Student's t tests and ANOVA.Differences were considered significant at P Ͻ 0.05 in two-tail distributions.

Results
Malignant PCC (16 sporadic PCC) had histologically confirmed liver metastases and elevated catecholamine levels during follow-up (Table 2), whereas locally invasive PCC (30 sporadic PCC) showed retroperitoneal soft tissue infiltration.Benign PCC (49 sporadic and 48 MEN 2A neoplasms) had no evidence of extraadrenal tumor growth and normal catecholamine levels during follow-up (Ͻ100 g/24 h).The tumors were found in 76 males ( 2A patients had either medullary thyroid carcinoma (36 cases) or C cell hyperplasia (12 cases).
Concordant TSG microsatellite patterns in both tumor compartments were observed in 110 PCC (76.9%;Fig. 2), and topographic genetic heterogeneity (LOH and/or somatic SNP) were found in 33 cases (23.1%;Fig. 2  PCC with topographic genetic heterogeneity showed significantly higher incidence of TP53 (P ϭ 0.00933) and NF1 (P Ͻ 0.00001) alterations at the peripheral compartment than topographically homogeneous PCC (Table 4).TP53 topographic heterogeneity (Tables 3 and 4) was found associated with other genetic alterations in malignant PCC, but as isolated genetic alterations in MEN 2A-related PCC (Fig. 3).NF1 microsatellite alterations were shown in 73.3% informative PCC (Table 4 and Figs. 2 and 3) and were the only TSG genetic alterations with significant differences between spo- All reactions were run with 1.5 mM of MgCl 2 , using 0.25 M of each primer and 50 M of each dNTP for the polymorphic regions of TSG tested.The PCR products were internally labeled with 0.3 Ci ␣͓ 32 P͔dCTP (3000 Ci/mmol, 10 mCi/ml, New England Nucleotide, Boston MA).All reactions were run in duplicate using 1 l of template.A long denaturation (4 min) was used in the first three cycles for each set of primers.The annealing temperature was 55 C for all primer sets (except for NF1, for which it was 52 C).The number of cycles was experimentally optimized to 26 for the polymorphic regions of TSG.radic and MEN 2A PCC (Table 5).NF1 abnormalities were also detected in tumors with topographic genetic heterogeneity in the case of sporadic PCC (eight of nine, 89%), whereas NF1 abnormalities were equally demonstrated in MEN 2A PCC regardless of the presence of topographic heterogeneity.No significant differences were observed for RB1 and WT1 loci.The comparison between sporadic and MEN 2A PCC revealed a significantly higher proportion of NF1 locus abnormalities (LOH-somatic SNP), so that the other TSG microsatellites differences were nonsignificant (Table 5).We found concordant TSG microsatellite pattern by compartments in 47 PCC (32.9%, 24 sporadic and 23 MEN 2A).Because the normal tissue LOH probability was 0.2 (10,11,(27)(28)(29)(30)(31), the probability of getting the same n loci involved in both peripheral and internal samples (two) would be (0.2 2 ) n .Under these circumstances, the probability of randomly finding concordant results in both PCC compartments (one TSG locus in 21 patients, two TSG loci in 10, and three TSG loci in 16) would be [(0.2 2 ) 1 ] 21 [(0.2 2 ) 2 ] 10 [(0.2 2 ) 3 ] 16 .Likewise, because the normal tissue ROH probability, P ROH ϭ 1 Ϫ P LOH ϭ1-0.2 ϭ 0.8 for a given marker, 0.8 5 for 5 markers, and (0.8 5 ) 2 for concordant peripheral and internal compartments, it will result in [(0.8 5 ) 2 ] 32 ϭ 9.75 ϫ 10 Ϫ32 for 32 PCC.
Immunostaining for mlh1/msh2 revealed statistically significant reduction of at least one of the proteins in the peripheral compartment of PCC with two or more TSG microsatellite abnormalities regardless of the histological FIG. 2. Microsatellite pathways in PCC.Microsatellite patterns; PCCs with concordant microsatellite pattern in internal and peripheral compartments.This homogeneous microsatellite pattern reveals a significant decrease in the intensity of the smaller allele of lanes 2 from both internal and peripheral PCC compartments (arrows).The allele pattern is the opposite of control (adrenal medulla) that shows higher intensity in the larger allele.The other tumor suppressor gene loci show no abnormalities.PCCs with intratumor heterogeneity and disconcordant microsatellite pattern in internal and peripheral compartments.Heterogeneous microsatellite patterns reveal a decreased intensity of the larger allele in lane 1 and additional allele bands in lane 3 (arrows) in the peripheral compartment.1, TP53(1); 2, TP53(2); 3, RB1; 4, WT1; and 5, NF1.NF1 locus microsatellite patterns.Lanes 1 (adrenal cortex) and 2 (adrenal medulla) correspond to two representative controls (from the same case shown in lanes 3 and 4), whereas lanes 3-12 show the microsatellite pattern in both peripheral (P) and internal (I) PCC compartments of five cases.Lanes 3 and 4 show concordant loss of the larger allele in both tumor compartments (compared with their matched controls in lanes 1 and 2).Lanes 5-12 show a discordant allele pattern in peripheral and internal compartments.The TSG microsatellite profile is shown.Malignant PCC (12 of 16, 75%) reveal two or more TSG loci with microsatellite abnormalities in the peripheral compartment, whereas all locally invasive and benign PCC show a lower incidence of microsatellite lesions.Mismatch protein expression is shown.Nuclear mlh1 and msh2 expression is demonstrated in PCC in the microsatellite-stable pathway, and at least one of these proteins was absent (in particular, mlh1) in PCC, showing microsatellite instability.MLH1 and MSH2 exon sequencing is shown.A normal sequence is demonstrated for these genes regardless of the microsatellite pattern.We are currently performing promoter methylation assays for MLH1.diagnosis (Fig. 2).No significant difference was observed in the mlh1/msh2 immunoexpression in PCC with less than two TSG genetic abnormalities (MS stable or MS instable low), but revealed a deficient MMR system at the peripheral compartment.Normal MLH1/MSH2 exons sequences were observed in all PCC analyzed regardless of immunoexpression and microsatellite status (Fig. 2).

Discussion
PCCs revealed two types of TSG microsatellite patterns: 1) PCC with no evidences of TSG microsatellite abnormalities (44.1%), and 2) PCC with microsatellite alterations (55.9%), probably resulting from multistep tumorigenesis and topographic clonal selection at the peripheral compartment through somatic topographic down-regulation of MMR proteins.Both accumulation of microsatellite lesion accumulation and intratumor heterogeneity characterize malignant PCC, in contrast to locally invasive PCC, which frequently reveals single locus alterations, especially involving NF1.
A PCC subgroup showed no demonstrable TSG alteration in tested introns (32 PCC, 43.8%).We previously optimized the DGGE protocol with appropriate controls, including positive, negative, and sensitivity (data not shown); the progressive dilution of a known positive case in a background of germline DNA gave us a sensitive threshold of 1% for a positive detection.We systematically microdissected at least 100 cells from each tumor compartment, a technique that would only miss the positive results from the DNA equivalent to less than one cell, which is probably clinically irrelevant.Normal tissue contamination could dilute the mutated DNA below the detection threshold, but this possibility was excluded by consistently negative results after careful microdissections.Although these TSG can show alterations in other introns, the low probability of finding no TSG alterations suggests that nonmalignant and sporadic PCC evolve through alternative molecular pathways (5,7,32,33).
The topographic intratumor heterogeneity suggests a differential selection of tumor cells by compartments, but can also be the expression of either selective clonal evolution or a simple passive byproduct of genetic instability (10,13,14,34).The differential kinetic profiles of topographic tumor compartments revealed lower cell turnover and apoptosis down-regulation in deep/peripheral compartments, resulting in accumulation of genetic alterations and segregation of tumor cells with differential genetic backgrounds, as demonstrated in colon and bladder (13,20,35).In these organs (as in the present study), the process has been linked with MMR protein down-regulation, and it is unlikely to be related to hypoxia, which is more pronounced in central compartments.However, the coexistence of genetic alterations in malignant PCC supports a key role in tumorigenesis, the topographic heterogeneity resulting from the accumulation of genetic damage in TP53 and NF1 loci.TP53 abnormalities were more frequently observed in topographically heterogeneous and malignant PCC.Krijger et al. (36) reported p53 immunoexpression between 10 -50% in most malignant PCC, supporting TP53 heterogeneity regardless of the genetic background (sporadic or familial) (5,6).The lower sensitivity of immunohistochemistry and the heterogeneous tissue distribution would explain the absence of significant immunoexpression differences.We found similar topographic heterogeneity in muscle-invasive transitional cell carcinoma of the urinary bladder, where TP53 abnormalities tended to concentrate in the deep tumor compartment, suggesting that it can represent the consequence of tumor cell selection (10,13,20).
NF1 microsatellite alterations were significantly more frequent in PCC with topographic heterogeneity and were the only markers differentiating sporadic from MEN 2A tumors.Reduced or absent NF1 gene expression (both mRNA and protein) has been previously documented in one of four sporadic PCC, three of 10 tumors from MEN 2A patients, and two of four tumors from patients with MEN2B, most of them expressing predominantly the type 1 NF1 isoform (37).These findings together support Knudson's two-mutation theory and the importance of the NF1 gene in PCC tumorigenesis in patients without neurofibromatosis (9, 38 -40), especially those with MEN syndromes [seven of 13 (53.8%) of our informative MEN 2A patients showed NF1 locus abnormalities].The NF1 gene product has an effect on RAS inhibition; this protein is expressed at the highest levels in immature and proliferating cells (41).The absence of NF1 inhibitory effects will favor increased cell proliferation, frequent presence of tetraploid cells, and, eventually, tumorigenesis (24,42).
The accumulation of TSG genetic lesions supports a monoclonal origin of tumors and expresses molecular tumor progression (10,11), contributing to the relatively high incidence of NF1 locus abnormalities found in MEN 2A patients (seven of 13 informative cases, 53.8%).Those results strongly support a homogeneous cell selection and a clonal origin of this PCC subset through a pathway that targets the evaluated TSG.This convergent tumor cell selection ends in dominant clones sharing genetic abnormalities, explaining the utility of coexistent LOH as a clonality marker (11).Therefore, clonality would be both the origin and the byproduct of neoplastic transformation (10,14,43).Tumor initiation and/or progres-sion in PCC might involve multiple genes apart from the RET oncogene (44).The MEN 2A kindred of this series carried RET germline mutation in codon 634 (45) and had coexistent thyroid lesions (18 medullary carcinoma and six C cell hyperplasia).Recently, RET mutations in codon 768 were found to segregate with medullary thyroid carcinomas or C cell hyperplasia only (46), and variable mechanisms are responsible for RET allelic imbalance (homozygosity, hemizygosity, and poly/monosomy), suggesting that multiple factors contribute to tumor development (47,48).X-Chromosome inactivation assays in patients from this kindred revealed the same X-chromosome inactivated in C cell hyperplasia foci from each lobe and in different nodules from adrenal medullary hyperplasias in a given patient (24,49).Those findings would be explained by early clonal expansions of both C cell and adrenal medullary precursors, which result in neoplasms when other genes are targeted and genetic alterations accumulate (18 of 24 MEN 2A PCC, 75.0%, had at least one TSG microsatellite abnormality).
The accumulation of TSG microsatellite abnormalities in the peripheral compartment correlates with the lack of mlh1/ msh2 immunoexpression and normal gene sequences (50).Replication error-positive samples have been reported in 30.8% of heterozygous PCC, the instability being the result of impaired cellular MMR (51), which leads to mutation accumulation in every cellular division.These findings express tumor progression with decreased aneuploid cell prevalence and loss of physiological cell kinetic correlations.The data suggest that molecular mechanisms of genomic instability are not necessarily independent and may not be fully defined by either microsatellite or chromosomal instability pathways; a subgroup of tumors showed no evidence of alterations in either of these two pathways of genomic instability (13,52).
MMR proteins normally identify and correct mismatched DNA sequences that can occur during DNA replication (11,53,54).Tumor progression in peripheral compartments may be the result of MMR protein down-regulation, which would contribute to: 1) lower DNA indices and the decreased prevalence of aneuploid cell lines detected in microsatellite-unstable neoplasms (55,56), and deep compartments of sporadic colorectal carcinomas (2,57,58); and 2) tumor cell heterogeneity, genetic instability, and biological progression.Because of intratumoral heterogeneity, at least two samples from each tumor should be screened (11), preferably from internal and peripheral compartments to cover the topographic tumor heterogeneity described in sporadic colorectal carcinomas (59) and muscle-invasive transitional cell carcinomas of the bladder (13).
In conclusion, the accumulation of TSG microsatellite alterations supports a convergent genetic selection and multistep tumorigenesis in sporadic and MEN 2A PCC.This selection process also expresses intratumoral heterogeneity with accumulation of microsatellite abnormalities at the PCC peripheral compartment, multiple loci, and TP53 for malignant PCC and NF1 for MEN 2A and locally invasive PCC.A PCC subset (nonmalignant and sporadic) with no TSG microsatellite abnormalities evolves through an alternative pathway.
FIG.1.PCC were analyzed by topographic compartments.The peripheral compartment comprised the 2.5-mm width tissue next to the transition between the tumor and the surrounding gland, whereas the internal compartment was the remaining tumor tissue inner to the peripheral rim.The same areas from these compartments were separately evaluated regarding microsatellites profile, sequencing, and immunoexpression of mlh1 and msh2.The brown dots in the inset represent the microscopic fields examined for the mlh-1 and msh-2 immunoexpression evaluations.

FIG. 3 .
FIG. 3. Malignant PCC frequently show topographic accumulation of TSG abnormalities at the peripheral tumor compartment (A) and simultaneous alterations of multiple TSG (B), especially involving TP53.

TABLE 1 .
Primer sequences and PCR cycling conditions for the amplification of polymorphic DNA regions of TSGs

TABLE 2 .
Clinicopathological features of malignant PCC a See Figs.2 and 3for details.

TABLE 3 .
Pheochromocytomas with topographic heterogeneity in microsatellite pattern of TSGs a a Genes showing different microsatellite pattern in the peripheral and internal compartments are highlighted in italics.bTwo samples were systematically evaluated from the peripheral (P) and internal (I) PCC compartments.cGene intron results were categorized as ROH, LOH, and SNP involving either the larger allele (L) or the smaller allele (S).Samples with only one allele band were considered noninformative (NI).dPCC types, Associated to MEN-2A (benign) and sporadic (benign, locally invasive, and malignant).

TABLE 4 .
Distribution of genetic abnormalities in tumor suppressor genes by topographic compartments in pheochromocytomas Pheochromocytomas were classified according to the microsatellite (MS) pattern of tumor suppressor genes in tumors revealing peripheral heterogeneity (more genetic alterations in the peripheral compartment) and tumors with identical microsatellite pattern in both compartments.bTumorsuppressor gene results were categorized in ROH, and LOH-SNP.cTable shows row percentage. a

TABLE 5 .
Distribution of genetic alterations in tumor suppressor genes in sporadic and MEN 2A pheochromocytomas Tumor suppressor gene results were categorized in ROH and LOH-SNP.
a b Table shows row percentage.