Fax: (317) 274-5346
Clonal divergence and genetic heterogeneity in clear cell renal cell carcinomas with sarcomatoid transformation
Version of Record online: 26 JUL 2005
Copyright © 2005 American Cancer Society
Volume 104, Issue 6, pages 1195–1203, 15 September 2005
How to Cite
Jones, T. D., Eble, J. N., Wang, M., MacLennan, G. T., Jain, S. and Cheng, L. (2005), Clonal divergence and genetic heterogeneity in clear cell renal cell carcinomas with sarcomatoid transformation. Cancer, 104: 1195–1203. doi: 10.1002/cncr.21288
- Issue online: 31 AUG 2005
- Version of Record online: 26 JUL 2005
- Manuscript Accepted: 25 APR 2005
- Manuscript Received: 15 MAR 2005
- renal cell carcinoma;
- sarcomatoid transformation;
- loss of heterozygosity;
- genetic heterogeneity;
- X-chromosome inactivation
Approximately 5% of clear cell renal cell carcinomas contain components with sarcomatoid differentiation. It has been suggested that the sarcomatoid elements arise from the clear cell tumors as a consequence of clonal expansions of neoplastic cells with progressively more genetic alterations. Analysis of the pattern of allelic loss and X-chromosome inactivation in both the clear cell and sarcomatoid components of the same tumor allows assessment of the genetic relationship of these tumor elements.
The authors of the current study examined the pattern of allelic loss in clear cell and sarcomatoid components of renal cell carcinomas from 22 patients who had tumors with both components. DNA samples were prepared from formalin-fixed, paraffin-embedded renal tissue sections using laser-capture microdissection. Five microsatellite polymorphic markers for putative tumor suppressor genes on 5 different chromosomes were analyzed. These included D3S1300 (3p14), D7S522 (7q31), D8S261 (8p21), D9S171 (9p21), and TP53 (17p13). In addition, X-chromosome inactivation analysis was performed in 14 tumors from female patients.
The clear cell components showed loss of heterozygosity (LOH) at the D3S1300, D7S522, D8S261, D9S171, and TP53 loci in 18% (4/22), 18% (4/22), 50% (10/20), 15% (3/20), and 20% (4/20) of informative cases, respectively. LOH in the sarcomatoid components was seen at the D3S1300, D7S522, D8S261, D9S171, and TP53 loci in 18% (4/22), 41% (9/22), 70% (14/20), 35% (7/20), and 20% (4/20) of informative cases, respectively. Six cases demonstrated an LOH pattern in the clear cell component that was not seen in the sarcomatoid component. Different patterns of allelic loss were seen in the clear cell and sarcomatoid components in 15 cases. Clonality analysis showed the same pattern of nonrandom X-chromosome inactivation in both clear cell and sarcomatoid components in 13 of the 14 cases studied. One case showed a random pattern of X-chromosome inactivation.
X-chromosome inactivation analysis data suggest that both clear cell and sarcomatoid components of renal cell carcinomas are derived from the same progenitor cell. Different patterns of allelic loss in multiple chromosomal regions were observed in clear cell and sarcomatoid components from the same patient. This genetic heterogeneity indicates genetic divergence during the clonal evolution of renal cell carcinoma. Cancer 2005. © 2005 American Cancer Society.
Sarcomatoid differentiation within carcinomas is common. In addition to its frequent occurrence in renal cell carcinoma (RCC),1, 2 it has been demonstrated in many other types of cancer, including head and neck squamous cell carcinoma,3 cancer of the upper respiratory tract,4 and cancers of the prostate5 and urinary bladder.6 Five to 10% of RCCs have areas of sarcomatoid morphology.1, 2, 7, 8 RCCs with sarcomatoid elements are characterized by highly malignant behavior2, 9 with increased local invasion and distant metastasis and have higher proliferative activity than other RCC types.10
Whereas specific types and subtypes of renal tumors are characterized by certain molecular and cytogenetic abnormalities,11 the genetic events associated with the progression of clear cell renal cell carcinoma to a sarcomatoid lesion are poorly understood. Carcinogenesis involves multiple genetic changes such as the activation of dominantly acting oncogenes that promote cell proliferation and the loss of tumor suppressor genes that negatively regulate cell proliferation. The inactivation of tumor suppressor genes by mutation or by loss or replacement of a chromosomal segment containing the allele provides a selective advantage essential for transformation or progression of a malignancy.12–15 Molecular and cytogenetic studies of renal cell carcinoma have shown a link between loss of tumor suppressor genes at chromosome 3p and tumor development.16–19 The high frequency of allelic loss at this and other chromosomal sites suggests that inactivation of tumor suppressor genes may be an important mechanism in renal cell carcinogenesis and tumor progression. Because the molecular and clonal relations of clear cell and sarcomatoid components of renal cell carcinomas is unknown, the authors of the current study undertook analysis of loss of heterozygosity (LOH) and X-chromosome inactivation status in both tumor components to define their relation.
MATERIALS AND METHODS
Seventeen women and 8 men with renal cell carcinoma underwent nephrectomy (n = 22) at the Indiana University School of Medicine (Indianapolis, IN) and Case Western Reserve University (Cleveland, OH) from 1995 to 2002. Patients had a mean age of 57 years (range, 36–77 yrs). All patients had both clear cell and sarcomatoid components in their renal cell carcinomas. All tumors were given a Fuhrman Grade 4 based on the presence of sarcomatoid elements. Pathologic staging was performed according to the 2002 TNM (tumor, lymph node, metastasis) classification system.20 Other clinical information and pathologic stages are shown in Table 1.
Tissue Samples and Microdissection
Archival materials from 22 cases of renal cell carcinoma (14 female patients and 8 male patients) with both clear cell and sarcomatoid components acquired from 1995 to 2002 were retrieved from the surgical pathology files of Indiana University Hospital and University Hospitals of Cleveland. Histologic sections were prepared from formalin-fixed, paraffin-embedded tissue and were stained with hematoxylin and eosin (H & E) for microscopic evaluation. From these slides, the clear cell and sarcomatoid components of the RCCs were identified (Fig. 1). Laser-assisted microdissection of the two components was performed (Fig. 1) on the unstained sections using a PixCell II Laser Capture Microdissection (LCM) system (Arcturus Engineering, Mountain View, CA), as previously described.21–24 Approximately 400 to 1000 cells of each component were microdissected from the 5-μm histologic sections. Normal tissue from each case was microdissected as a control.
Amplification of DNA
The dissected cells were deparaffinized with xylene and ethyl alcohol. Polymerase chain reaction (PCR) was used to amplify genomic DNA at various specific loci on five different chromosomes: 3p14 (D3S1300), 7q31 (D7S522), 8p21 (D8S261), 9p21 (D9S171), and 17p13 (TP53). Previous studies demonstrated that (LOH) at these loci occurs frequently in renal cell carcinomas.25–39 The tumor-suppressor gene FHIT (fragile histidine triad) locus is present at 3p14 (D3S1300). The TP53 locus corresponds to the gene encoding the p53 protein. Mutations of the p53 gene are the most common genetic abnormalities in cancer.40 D9S171 includes regions of the putative tumor-suppressor gene p16. The remaining loci also correspond to putative tumor-suppressor genes. Polymerase chain reaction (PCR) amplification and gel electrophoresis were performed as previously described.41–45 The criterion for allelic loss was complete or nearly complete absence of one allele in tumor DNA.41–43, 46–48 PCRs for each polymorphic microsatellite marker were repeated at least twice from the same DNA preparations, and the same results were obtained. Results were reported as noninformative when visual inspection could not distinguish two distinct band forms in control DNA following PCR amplification.
Detection of X-Chromosome Inactivation
X-chromosome inactivation analysis was performed on the renal cell carcinomas from the 14 female patients. DNA samples were prepared from the clear cell and sarcomatoid tumor components from the same patient. The dissected cells were placed in 15 μL of buffer (i.e., 10 mM Tris-HCl, 1 mM EDTA, 1% Tween 20, and 0.2 mg/mL of proteinase K (pH 8.3) and incubated overnight at 37 °C. The solution was boiled for 10 minutes to inactivate the proteinase K and used directly for subsequent clonal analysis without further purification. Aliquots (8 μL) of the DNA extract were digested overnight at 37 °C with 1 U of HhaI restriction endonuclease (New England Biolabs Inc., Beverly, MA) in a total volume of 10 μL. Equivalent aliquots of the DNA extracts were also incubated in the digestion buffer without HhaI endonuclease as control reactions for each sample. After the incubation, 3 μL of digested or nondigested DNA was amplified in a 25-μL PCR volume containing 0.1 μL32[P]α-labeled deoxyadenosine triphosphate (dATP) (3000 Ci/mmol), 4 μM AR-sense primer (5′TCC AGA ATC TGT TCC AGA GCG TGC3′), 4 μM AR-antisense primer (5′GCT GTG AAG GTT GCT GTT CCT CAT3′), 4% dimethyl sulfoxide, 2.5 mM MgCl2, 300 μM deoxycytidine triphosphate, 300μM deoxythymidine triphosphate, 300 μM deoxyguanosine triphosphate, 300 μM dATP, and 0.13 U Taq DNA polymerase (Perkin-Elmer Corp., Norwalk, CT). Each PCR amplification had an initial denaturation step of 95 °C for 8 minutes, followed by 32 cycles at 95 °C for 40 seconds, at 63 °C for 40 seconds, and at 72 °C for 60 seconds, and then followed by a single final extension step at 72 °C for 10 minutes. The PCR products were then diluted with 4 μL of loading buffer containing 95% formamide, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol FF (Sigma Chemical Co., St. Louis, MO). The samples were heated to 95 °C for 5 minutes and then placed on ice. Three microliters of the reaction mixture was loaded onto 6.5% polyacrylamide-denaturing gels without formamide, and the PCR products were separated by electrophoresis at 1600 V for 4–7 hours. The bands were observed after autoradiography with Kodak X-OMAT film (Eastman Kodak Company, Rochester, NY) for 8–16 hours.
Analysis of X-Chromosome Inactivation
Cases were considered to be informative if two AR allelic bands were detected after PCR amplification in normal control samples that had not been treated with HhaI. Only informative cases (i.e., those without a skewed pattern of X-chromosome inactivation after being treated with HhaI in normal control samples) were included in the analysis. In tumor samples, nonrandom X-chromosome inactivation was defined as a complete or a nearly complete absence of an AR allele after HhaI digestion, which indicated a predominance of one allele.44, 45, 49, 50 Tumors were considered to be of the same clonal origin if the same AR allelic inactivation pattern was detected in both the clear cell and sarcomatoid components of the renal tumor. Tumors were considered to be of independent origin if alternate predominance of AR alleles after HhaI digestion (different allelic inactivation patterns) was detected in the two components of the tumor.
Twenty of the 22 (91%) renal cell carcinomas containing clear cell and sarcomatoid components showed allelic loss in one or both of these components (Table 1). The number of specific loci lost ranged from one to four. Nearly all of the 22 tumors demonstrated a different pattern of allelic loss. Thirteen of 22 clear cell components and 20 of 22 sarcomatoid components showed allelic loss in at least 1 of the 5 microsatellite polymorphic markers (Fig. 2).
Fifteen of the 22 tumors demonstrated different patterns of allelic loss in the clear cell and sarcomatoid components. Five tumors demonstrated an identical LOH pattern in the clear cell and sarcomatoid components. Two tumors did not show LOH at any of the five loci examined in either component (Table 1).
The frequency of allelic loss in the clear cell components of informative cases was 18% (4 of 22) with D3S1300; 18% (4 of 22) with D7S522; 50% (10 of 20) with D8S261; 15% (3 of 20) with D9S171; and 20% (4 of 20) with TP53. The frequency of allelic loss in the sarcomatoid components of informative cases was 18% (4 of 22) with D3S1300; 41% (9 of 22) with D7S522; 70% (14 of 20) with D8S261; 35% (7 of 20) with D9S171; and 20% (4 of 20) with TP53. Figure 3 shows the LOH frequency of the two renal cell carcinoma components at each of the five loci studied.
The allelic loss patterns at the five loci examined were quite variable in both components. Six cases demonstrated an LOH pattern in the clear cell component that was not seen in the sarcomatoid component.
Clonality analysis showed the same pattern of nonrandom X-chromosome inactivation in both clear cell and sarcomatoid components in 13 of the 14 cases that were informative, suggesting a common clonal origin for the two components. One case showed a random pattern of X-chromosome inactivation (Case 3).
The purpose of the current study was to investigate the genetic relation between sarcomatoid and clear cell components within renal cell carcinomas comprised of these elements. Our findings support the hypothesis that the sarcomatoid elements seen in conventional renal cell carcinomas arise from the clear cell population as a consequence of clonal expansions of neoplastic cells with progressively greater genetic alterations.51 The differing allelic loss patterns displayed by the two neoplastic components suggests that intratumoral heterogeneity exists in these cancers and reflects genetic divergence during the clonal evolution of renal cell carcinomas. Our X-chromosome inactivation data suggest that both tumor components are derived from the same progenitor cell.
Loss of heterozygosity (LOH) has been found at various chromosomal loci in many types of human cancers,52 including renal cell carcinomas.25–39 The chromosomal regions where LOH has been detected are thought to contain specific genes, the disruption of which leads to either neoplastic transformation or progression.15 The various stages of neoplastic progression are frequently accompanied by histologic evidence of cellular dedifferentiation or transformation with subpopulations of higher grade cells growing within lower grade lesions.51 This phenomenon is clearly demonstrable in renal cell carcinomas on histologic sections, as morphologic heterogeneity in these neoplasms is characterized by the presence of clear cell and sarcomatoid elements. The results of our X-chromosome inactivation analysis provide strong evidence for a common progenitor cell origin for both the clear cell and sarcomatoid components. However, LOH results highlight the significant genetic heterogeneity seen within renal cell carcinomas with both components. Based on these data, it is unlikely that genes at any of the loci studied are essential to tumorigenesis or progression. Nearly every tumor displayed an unique allelic loss pattern, and no locus was affected in every case. Although molecular heterogeneity clearly is present, our data do not provide sufficient evidence to establish a multistep model for neoplastic transformation and progression in RCCs, such as that seen in colorectal adenocarcinoma.53–56 A single route for RCC progression may not exist. Future studies analyzing allelic loss at additional loci may help to determine whether a true model of tumor progression can be established.
The most consistently informative marker of the clonal composition of neoplastic disorders in females is the nonrandom pattern of X-chromosome inactivation.44, 45, 57, 58 Based on our X-chromosome inactivation data, we conclude that the clear cell and sarcomatoid components in renal cell carcinomas comprised of these elements share a common pathway of tumorigenesis despite their conspicuous divergence at the phenotypic level. The same pattern of nonrandom X chromosome inactivation was seen in both the clear cell and the sarcomatoid components in 13 of the 14 cases that were informative, suggesting a common clonal origin for the two components. The one exceptional case (Case 3) showed a random pattern of X-chromosome inactivation in both the clear cell and sarcomatoid elements. Results for this case are shown in Figure 2. Several studies have shown that random X-chromosome inactivation may be observed in up to 50% of invasive cancers.59–63 For example, in a study by Buller et al., nonrandom X-chromosome inactivation was observed in only 53% of patients with invasive ovarian carcinoma.59 There are a number of possible explanations for the persistence of biallelic bands after digestion by a methylation-sensitive restriction enzyme in tumor samples (clonal cell populations). These include: 1) incomplete digestion of DNA samples prepared from formalin-fixed, paraffin-embedded tissues, 2) contamination of normal tissues, 3) the presence of X-chromosome aneuploidy, 4) the coexistence of multiple tumor subclones of independent origins, 5) reactivation of inactive X-chromosome–linked genes,64, 65 and 6) variable methylation patterns at the CpG sites of the androgen receptor locus. Some other mechanisms may attribute to the inactive status of X chromosome in females.65–69
In conclusion, our LOH and X-chromosome inactivation data support the contention that both clear cell and sarcomatoid tumor components in renal cell carcinomas arise from a common cell of origin and that clonal divergence occurs during tumor progression with subpopulations of neoplastic cells acquiring unique genetic alterations. This genetic divergence following the initial neoplastic transformation is reflected in the molecular heterogeneity seen in the LOH analysis.
- 20AJCC Cancer Staging Manual, 6th ed. New York: Springer-Verlag, 2002., , , , , , et al.