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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-1645
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 3 1150-1158
Copyright © 2006 by The Endocrine Society

Topographic Molecular Profile of Pheochromocytomas: Role of Somatic Down-Regulation of Mismatch Repair

Alfredo Blanes, Juan J. Sanchez-Carrillo and Salvador J. Diaz-Cano

Department of Pathology, University of Malaga School of Medicine (A.B., J.J.S.-C., S.J.D.-C.), Malaga E29010, Spain; and Department of Pathology, King’s College Hospital and King’s College School of Medicine (S.J.D.-C.), London SE5 9RS, United Kingdom

Address all correspondence and requests for reprints to: Dr. Salvador J. Diaz-Cano, Department of Histopathology, King’s College Hospital, Denmark Hill, London SE5 9RS, United Kingdom. E-mail: salvador.diaz-cano{at}kcl.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
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: Pheochromocytomas were classified as malignant (16 sporadic tumors with distant metastases), locally invasive (30 sporadic tumors showing retroperitoneal 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 involved TP53 in 40 of 134 informative cases (29.9%), RB1 in 22 of 106 informative cases (20.8%), WT1 in 32 of 120 informative cases (26.7%), and NF1 in 32 of 80 informative cases (40.0%). 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 < 0.001), whereas locally invasive pheochromocytomas showed a significantly higher incidence of NF1 alterations (P < 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 pheochromocytomas, suggesting a multistep tumorigenesis through somatic topographic down-regulation of MMR proteins. Locally invasive pheochromocytomas reveal topographic heterogeneity and single-locus microsatellite alterations, especially involving NF1.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
PHEOCHROMOCYTOMAS (PCC) ARE heterogeneous tumors (1, 2, 3, 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, 6, 7, 8, 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, 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 G1-S transition (TP53, RB1), RAS pathway (NF1), and development (WT1).


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Case selection

Sporadic (n = 95) and MEN 2A (n = 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 (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 (~0.4 mm2) from two 20-µm unstained paraffin sections/compartment (Fig. 1Go). Appropriate controls were included for each test (adrenal medulla, adrenal cortex, and periadrenal soft tissue).


Figure 1
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  		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.

 
DNA was extracted using a modified phenol-chloroform protocol, precipitated with ice-cold absolute ethanol, and resuspended in 10 µl Tris-HCl buffer (pH 8.4) (18). DNA was then used for PCR amplification of TSG intron microsatellites (Table 1Go) (13, 19). The tests were run in a PerkinElmer thermal cycler model 480 (PerkinElmer, Norwalk, CT). The entire 10-µl PCR volume was electrophoresed into 8% denaturing gradient polyacrylamide gels; dried gels were put inside developing cassettes containing one intensifying screen and preflashed films (Kodak XAR, Eastman Kodak, Inc., Rochester, NY) (13, 19, 20). The radiographs were developed using an automated processor Kodak O-MAT 100 (Eastman Kodak Co.).


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

 
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 µl 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 µg/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 3,3'-diaminobenzidine tetrahydrochloride with 0.3% H2O2 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 mm2) 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 < 0.05 in two-tail distributions.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Malignant PCC (16 sporadic PCC) had histologically confirmed liver metastases and elevated catecholamine levels during follow-up (Table 2Go), 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 (<100 µg/24 h). The tumors were found in 76 males (51 sporadic and 25 MEN 2A) and 67 females (44 sporadic and 23 MEN 2A), and all MEN 2A patients had either medullary thyroid carcinoma (36 cases) or C cell hyperplasia (12 cases).


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  		TABLE 2. Clinicopathological features of malignant PCC

 
Microsatellite alterations were observed in 80 PCC (55.9%; 45 sporadic and 35 MEN 2A). TSG microsatellite analysis revealed TP53 alterations in 39 of 131 informative cases (29.8%, screening two introns), RB1 abnormalities in 22 of 104 informative cases (21.2%), WT1gene lesions in 31 of 118 informative cases (26.3%), and NF1 alterations in 31 of 78 informative cases (39.7%). No TSG microsatellite abnormalities were revealed in 63 PCC (44.1%, only two metastatic). The same microsatellite locus was involved in both compartments of 22 PCC (15.4%): TP53 in 10 benign cases (7.0%) and WT1 in 12 cases (8.4%, only two metastatic). Microsatellite abnormality in one TSG in the peripheral compartment only was observed involving NF1 locus in 14 nonmetastatic PCC (9.8%), TP53 in four benign PCC (2.8%), and WT1 locus in two benign PCC (1.4%). The remaining PCC had two TSG loci altered in 20 cases (14.0%, eight malignant and 12 benign PCC) and three TSG loci in 20 cases (14.0%, four of them metastatic). Mutations were confirmed by sequencing of the corresponding introns of all cases with microsatellite abnormalities (Fig. 2Go), but were not detected in control tissues, confirming their somatic nature.


Figure 2
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  		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.

 
Concordant TSG microsatellite patterns in both tumor compartments were observed in 110 PCC (76.9%; Fig. 2Go), and topographic genetic heterogeneity (LOH and/or somatic SNP) were found in 33 cases (23.1%; Fig. 2Go and Table 3Go): 12 benign, nine locally invasive, and 12 malignant. PCC with discordant microsatellite pattern revealed more TSG loci involved in the peripheral compartment: NF1 locus in 16 nonmetastatic PCC (11.2%), TP53 in six benign PCC (4.2%), and variable combinations of TP53-RB1-WT1-NF1 in 12 malignant PCC (8.4%; Table 3Go). PCC with concordant microsatellite pattern of TSG showed demonstrable genetic alterations in at least one TSG locus in 47 cases (32.9%, only two of them malignant). Finally, a subset of PCC (63 cases, 44.1%, two malignant only) revealed no alterations in the TSG loci analyzed.


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  		TABLE 3. Pheochromocytomas with topographic heterogeneity in microsatellite pattern of TSGs1

 
PCC with topographic genetic heterogeneity showed significantly higher incidence of TP53 (P = 0.00933) and NF1 (P < 0.00001) alterations at the peripheral compartment than topographically homogeneous PCC (Table 4Go). TP53 topographic heterogeneity (Tables 3Go and 4Go) was found associated with other genetic alterations in malignant PCC, but as isolated genetic alterations in MEN 2A-related PCC (Fig. 3Go). NF1 microsatellite alterations were shown in 73.3% informative PCC (Table 4Go and Figs. 2Go and 3Go) and were the only TSG genetic alterations with significant differences between sporadic and MEN 2A PCC (Table 5Go). 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 5Go).


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  		TABLE 4. Distribution of genetic abnormalities in tumor suppressor genes by topographic compartments in pheochromocytomas

 

Figure 3
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  		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.

 

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  		TABLE 5. Distribution of genetic alterations in tumor suppressor genes in sporadic and MEN 2A pheochromocytomas

 
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 (0.22)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 [(0.22)1]21[(0.22) 2]10[(0.22)3]16. Likewise, because the normal tissue ROH probability, PROH = 1 – PLOH=1–0.2 = 0.8 for a given marker, 0.85 for 5 markers, and (0.85)2 for concordant peripheral and internal compartments, it will result in [(0.85)2]32 = 9.75 x 10–32 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 diagnosis (Fig. 2Go). 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. 2Go).


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
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, 39, 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 progression 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.


   Acknowledgments
 
This work was performed at the Department of Pathology, Tufts University-New England Medical Center (Boston, MA) and Barts and The London National Health Service Trust (London, UK).


   Footnotes
 
First Published Online January 4, 2006

Abbreviations: DGGE, Denaturing gradient gel electrophoresis; ISEL, in situ end labeling; LOH, loss of heterozygosity; MEN 2A, multiple endocrine neoplasia type 2A; MMR, mismatch repair; NF1, neurofibromatosis 1; PCC, pheochromocytoma; RB1, retinoblastoma; ROH, retention of heterozygosity; SNP, single nucleotide polymorphism; TP53, tumor protein p53; TSG, tumor suppressor gene; WT1, Wilms tumor 1.

This paper was presented in part as abstracts at the Annual Meetings of United States and Canadian Academies of Pathology in Orlando, FL (March 1997), and San Antonio, TX (March 2005); the XXII International Congress of the International Academy of Pathology in Nice, France (October 1998); and the Pathological Society of Great Britain and Ireland, Maastricht 2001.

Received July 22, 2005.

Accepted December 27, 2005.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

  1. Neumann HP, Berger DP, Sigmund G, Blum U, Schmidt D, Parmer RJ, Volk B, Kirste G 1993 Pheochromocytomas, multiple endocrine neoplasia type 2, and von Hippel-Lindau disease. N Engl J Med [Erratum (1994) 331:1535] 329:1531–1538
  2. Lack EE 1997 Tumors of the adrenal gland and extra-adrenal paraganglia. Washington DC: Armed Forces Institute of Pathology
  3. Carney JA, Sizemore GW, Sheps SG 1976 Adrenal medullary disease in multiple endocrine neoplasia, type 1. Pheochromocytoma and its presursors. Am J Clin Pathol 66:279–290[Medline]
  4. Koch CA, Pacak K, Chrousos GP 2002 The molecular pathogenesis of hereditary and sporadic adrenocortical and adrenomedullary tumors. J Clin Endocrinol Metab 87:5367–5384[Abstract/Free Full Text]
  5. Khosla S, Patel VM, Hay ID, Schaid DJ, Grant CS, van Heerden JA, Thibodeau SN 1991 Loss of heterozygosity suggests multiple genetic alterations in pheochromocytomas and medullary thyroid carcinomas. J Clin Invest 87:1691–1699[Medline]
  6. Shin E, Fujita S, Takami K, Kurahashi H, Kurita Y, Kobayashi T, Mori T, Nishisho I, Takai S 1993 Deletion mapping of chromosome 1p and 22q in pheochromocytoma. Jpn J Cancer Res 84:402–408[CrossRef]
  7. Vargas MP, Zhuang Z, Wang C, Vortmeyer A, Linehan WM, Merino MJ 1997 Loss of heterozygosity on the short arm of chromosomes 1 and 3 in sporadic pheochromocytoma and extra-adrenal paraganglioma. Hum Pathol 28:411–415[CrossRef][Medline]
  8. Yokogoshi Y, Yoshimoto K, Saito S 1990 Loss of heterozygosity on chromosomes 1 and 11 in sporadic pheochromocytomas. Jpn J Cancer Res 81:632–638[CrossRef]
  9. Koch CA, Huang SC, Zhuang Z, Stolle C, Azumi N, Chrousos GP, Vortmeyer AO, Pacak K 2002 Somatic VHL gene deletion and point mutation in MEN 2A-associated pheochromocytoma. Oncogene 21:479–482[CrossRef][Medline]
  10. Diaz-Cano SJ 1998 Clonality studies in the analysis of the adrenal medullary proliferations: application principles and limitations. Endocr Pathol 9:301–316[Medline]
  11. Diaz-Cano SJ, Blanes A, Wolfe HJ 2001 PCR techniques for clonality assays. Diagn Mol Pathol 10:24–33[CrossRef][Medline]
  12. Sternlicht M, Mirell C, Safarians S, Barsky SH 1994 A novel strategy for the investigation of clonality in precancerous disease states and early stages of tumor progression. Biochem Biophys Res Commun 199:511–518[CrossRef][Medline]
  13. Diaz-Cano SJ, Blanes A, Rubio J, Matilla A, Wolfe HJ 2000 Molecular evolution and intratumor heterogeneity by topographic compartments in muscle-invasive transitional cell carcinoma of the urinary bladder. Lab Invest 80:279–289[Medline]
  14. Diaz-Cano SJ, Blanes A 1999 Influence of intratumor heterogeneity in the interpretation of marker results in pheochromocytomas. J Pathol 189:627–628[CrossRef][Medline]
  15. Smith HS 1990 Stochastic model for interpreting the data on loss of heterozygosity in breast cancer. J Natl Cancer Inst 82:793–794[Free Full Text]
  16. Kinzler KW, Vogelstein B 1996 Lessons from hereditary colorectal cancer. Cell 87:159–170[CrossRef][Medline]
  17. DeLellis RA, Wolfe HJ, Gagel RF, Feldman ZT, Miller HH, Gang DL, Reichlin S 1976 Adrenal medullary hyperplasia. A morphometric analysis in patients with familial medullary thyroid carcinoma. Am J Pathol 83:177–196[Abstract]
  18. Diaz-Cano SJ, Brady SP 1997 DNA extraction from formalin-fixed paraffin-embedded tissues: Protein digestion as a limiting step for retrieval of high quality DNA. Diagn Mol Pathol 6:342–346[CrossRef][Medline]
  19. Cawkwell L, Lewis FA, Quirke P 1994 Frequency of allele loss of DCC, p53, RBI, WT1, NF1, NM23 and APC/MCC in colorectal cancer assayed by fluorescent multiplex polymerase chain reaction. Br J Cancer 70:813–818[Medline]
  20. Blanes A, Rubio J, Martinez A, Wolfe HJ, Diaz-Cano SJ 2002 Kinetic profiles by topographic compartments in muscle-invasive transitional cell carcinomas of the bladder: role of TP53 and NF1 genes. Am J Clin Pathol 118:93–100[Abstract/Free Full Text]
  21. Mutter GL, Boynton KA 1995 X chromosome inactivation in the normal female genital tract: Implications for identification of neoplasia. Cancer Res 55:5080–5084[Abstract/Free Full Text]
  22. Diaz-Cano SJ, de Miguel M, Blanes A, Tashjian R, Galera H, Wolfe HJ 2000 Clonality as expression of distinctive cell kinetic patterns in nodular hyperplasias and adenomas of the adrenal cortex. Am J Pathol 156:311–319[Abstract/Free Full Text]
  23. Diaz-Cano SJ 2000 Designing a molecular analysis of clonality in tumours. J Pathol 191:343–344[CrossRef][Medline]
  24. Diaz-Cano SJ, de Miguel M, Blanes A, Tashjian R, Wolfe HJ 2001 Germline RET 634 mutation positive MEN 2A-related C-cell hyperplasias have genetic features consistent with intraepithelial neoplasia. J Clin Endocrinol Metab 86:3948–3957[Abstract/Free Full Text]
  25. Simpson JF, Dutt PL, Page DL 1992 Expression of mitosis per thousand cells and cell density in breast carcinomas. A proposal. Hum Pathol 23:608–611[CrossRef][Medline]
  26. Diaz-Cano SJ, Leon MM, de Miguel M, Galera-Davidson H, Wolfe HJ 1996 Mitotic index quantification: Different approaches and their value in adrenal cortical proliferative lesions. Lab Invest 74:170A (Abstract)
  27. Chen LC, Kurisu W, Ljung BM, Goldman ES, Moore DI, Smith HS 1992 Heterogeneity for allelic loss in human breast cancer. J Natl Cancer Inst 84:506–510[Abstract/Free Full Text]
  28. Deng G, Lu Y, Zlotnikov G, Thor AD, Smith HS 1996 Loss of heterozygosity in normal tissue adjacent to breast carcinomas. Science 274:2057–2059[Abstract/Free Full Text]
  29. Wolman SR, Heppner GH 1992 Genetic heterogeneity in breast cancer. J Natl Cancer Inst 84:469–470[Free Full Text]
  30. Sager R 1989 Tumor suppressor genes: The puzzle and the promise. Science 246:1406–1412[Abstract/Free Full Text]
  31. Diaz-Cano SJ, Wolfe HJ 1997 Clonality in Kaposi’s sarcoma. N Engl J Med 337:571–572[Medline]
  32. Woodward ER, Eng C, McMahon R, Voutilainen R, Affara NA, Ponder BA, Maher ER 1997 Genetic predisposition to phaeochromocytoma: analysis of candidate genes GDNF, RET and VHL. Hum Mol Genet 6:1051–1056[Abstract/Free Full Text]
  33. Moley JF, Brother MB, Fong CT, White PS, Baylin SB, Nelkin B, Wells SA, Brodeur GM 1992 Consistent association of 1p loss of heterozygosity with pheochromocytomas from patients with multiple endocrine neoplasia type 2 syndromes. Cancer Res 52:770–774[Abstract/Free Full Text]
  34. Pohlink C, Tannapfe A, Eichfeld U, Schmidt F, Fuhrer D, Paschke R, Koch CA 2004 Does tumor heterogeneity limit the use of the Weiss criteria in the evaluation of adrenocortical tumors? J Endocrinol Invest 27:565–569[Medline]
  35. Jimenez JJ, Blanes A, Diaz-Cano SJ 2003 Microsatellite instability in colon cancer. N Engl J Med 349:1774–1776; author reply 1774–1776[CrossRef][Medline]
  36. Krijger RR, van der Harst E, van der Ham F, Stijnen T, Dinjen WNM, Koper JW, Bruining HA, Lamberts SWJ, Bosman FT 1999 Prognostic value of p53, bcl-2, and c-erbB-2 protein expression in phaeochromocytomas. J Pathol 188:51–55[CrossRef][Medline]
  37. Gutmann DH, Geist RT, Rose K, Wallin G, Moley JF 1995 Loss of neurofibromatosis type I (NF1) gene expression in pheochromocytomas from patients without NF1. Genes Chromosomes Cancer 13:104–109[Medline]
  38. Knudson Jr AG 1971 Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci USA 68:820–823[Abstract/Free Full Text]
  39. Knudson Jr AG, Strong LC 1972 Mutation and cancer: neuroblastoma and pheochromocytoma. Am J Hum Genet 24:514–532[Medline]
  40. Huang SC, Koch CA, Vortmeyer AO, Pack SD, Lichtenauer UD, Mannan P, Lubensky IA, Chrousos GP, Gagel RF, Pacak K, Zhuang Z 2000 Duplication of the mutant RET allele in trisomy 10 or loss of the wild-type allele in multiple endocrine neoplasia type 2-associated pheochromocytomas. Cancer Res 60:6223–6226[Abstract/Free Full Text]
  41. Furth ME, Aldrich TH, Cordon-Cardo C 1987 Expression of ras proto-oncogene proteins in normal human tissues. Oncogene 1:47–58[Medline]
  42. Gonzalez-Campora R, Diaz Cano S, Lerma-Puertas E, Rios Martin JJ, Salguero Villadiego M, Villar Rodriguez JL, Bibbo M, Davidson HG 1993 Paragangliomas. Static cytometric studies of nuclear DNA patterns. Cancer 71:820–824[CrossRef][Medline]
  43. Alexander P 1985 Do cancers arise from a single transformed cell or is monoclonality of tumours a late event in carcinogenesis? Br J Cancer 51:453–457[Medline]
  44. Koch CA, Huang SC, Moley JF, Azumi N, Chrousos GP, Gagel RF, Zhuang Z, Pacak K, Vortmeyer AO 2001 Allelic imbalance of the mutant and wild-type RET allele in MEN 2A-associated medullary thyroid carcinoma. Oncogene 20:7809–7811[CrossRef][Medline]
  45. Gagel RF, Cote GJ, Martins Bugalho MJ, Boyd III AE, Cummings T, Goepfert H, Evans DB, Cangir A, Khorana S, Schultz PN 1995 Clinical use of molecular information in the management of multiple endocrine neoplasia type 2A. J Intern Med 238:333–341[Medline]
  46. Boccia LM, Green JS, Joyce C, Eng C, Taylor SA, Mulligan LM 1997 Mutation of RET codon 768 is associated with the FMTC phenotype. Clin Genet 51:81–85[Medline]
  47. Marx SJ 2005 Molecular genetics of multiple endocrine neoplasia types 1 and 2. Nat Rev Cancer 5:367–375[CrossRef][Medline]
  48. Spiegel AM 2004 Focus on hereditary endocrine neoplasia. Cancer Cell 6:327–332[CrossRef][Medline]
  49. Diaz-Cano SJ, de Miguel M, Blanes A, Tashjian R, Galera H, Wolfe HJ 2000 Clonal patterns in phaechromocytomas and MEN-2A adrenal medullary hyperplasias: histologic and kinetic correlates. J Pathol 192:221–228[CrossRef][Medline]
  50. Blanes A, Diaz-Cano SJ Sporadic colorectal carcinomas: are microsatellite tumor profile and mismatch repair defects equivalent? World J Surg, in press
  51. Pecina-Slaus N, Nikuseva-Martic T, Gall-Troselj K, Radic K, Hrascan R 2005 Replication error-positive samples found in pheochromocytomas. In Vivo 19:359–365[Abstract/Free Full Text]
  52. Goel A, Arnold CN, Niedzwiecki D, Chang DK, Ricciardiello L, Carethers JM, Dowell JM, Wasserman L, Compton C, Mayer RJ, Bertagnolli MM, Boland CR 2003 Characterization of sporadic colon cancer by patterns of genomic instability. Cancer Res 63:1608–1614[Abstract/Free Full Text]
  53. Duval A, Hamelin R 2002 Mutations at coding repeat sequences in mismatch repair-deficient human cancers: toward a new concept of target genes for instability. Cancer Res 62:2447–2454[Abstract/Free Full Text]
  54. Peltomaki P 2001 DNA mismatch repair and cancer. Mutat Res 488:77–85[CrossRef][Medline]
  55. Thibodeau SN, French AJ, Cunningham JM, Tester D, Burgart LJ, Roche PC, McDonnell SK, Schaid DJ, Vockley CW, Michels VV, Farr Jr GH, O’Connell MJ 1998 Microsatellite instability in colorectal cancer: different mutator phenotypes and the principal involvement of hMLH1. Cancer Res 58:1713–1718[Abstract/Free Full Text]
  56. Liang JT, Chang KJ, Chen JC, Lee CC, Cheng YM, Hsu HC, Chien CT, Wang SM 1999 Clinicopathologic and carcinogenetic appraisal of DNA replication error in sporadic T3N0M0 stage colorectal cancer after curative resection. Hepatogastroenterology 46:883–890[Medline]
  57. Jimenez-Martin JJ, Miranda MT, Blanes A, Diaz-Cano SJ 2003 Expression of mlh1/msh2 in colo-rectal adenocarcinomas: SAGE/microarray profile and tumor phenotype. Lab Invest 83:296A (Abstract)
  58. Blanes A, Jimenez-Martin JJ, Miranda MT, Diaz-Cano SJ 2003 Abscence of the physiologic cellular kinetic balance and down-regulation of mlh1/msh2 characterize deep topographic compartments of colo-rectal adenocarcinomas. Lab Invest 83:114A–115A (Abstract)
  59. Chapusot C, Martin L, Bouvier AM, Bonithon-Kopp C, Ecarnot-Laubriet A, Rageot D, Ponnelle T, Laurent Puig P, Faivre J, Piard F 2002 Microsatellite instability and intratumoural heterogeneity in 100 right-sided sporadic colon carcinomas. Br J Cancer 87:400–404[CrossRef][Medline]




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